Asp68His mutation in the A1 domain of human factor V causes impaired secretion and ineffective translocation.

Congenital factor V (FV) deficiency is a rare inherited disorder. We determined the mechanism of a missense mutation, Asp68His, in the A1 domain of the FV protein, is associated with severe FV deficiency. We characterized the mutant FV-Asp68His protein using in vitro expression studies by using specific secretion and degradation pathway inhibitors and analysed the intracellular translocation of the mutant protein by immunofluorescence staining. The Asp68His mutation caused very low levels of FV protein in the conditioned media, with normal specific FV activity. Similar mRNA degradation rates between FV-wild-type (wt) and FV-Asp68His mRNA showed that the Asp68His mutation does not affect FV expression at the transcriptional level. A specific secretion pathway inhibitor, brefeldin A, was used to demonstrate that the lower efficiency of transport to the outside of the cell for FV-Asp68His mutant protein compared with that of the FV-wt protein. Furthermore, we showed that the Asp68His mutation resulted in increased intracellular degradation through a MG132-mediated proteasomal degradation pathway. In the transfected cell lysates, FV-wt protein had multiple posttranslational modified forms, but the FV-Asp68His protein was not completely glycosylated. We further observed that the FV-Asp68His protein was retrieved in the endoplasmic reticulum only and did not undergo transport to the Golgi apparatus, leading to impaired secretion. These results strongly suggest that the Asp68His mutation may result in intracellular defective trafficking and enhanced degradation, and impaired secretion of FV protein.

Metabolic syndrome and insulin resistance in relation to biliary tract cancer and stone risks: a population-based study in Shanghai, China.

BACKGROUND: Serum lipids, diabetes, and obesity, individual components of metabolic syndrome, are associated with biliary tract cancer and stone risk, but the associations of metabolic syndrome or insulin resistance with biliary tract cancers and stones are not well studied. METHODS: In this population-based case-control study in Shanghai, China (627 biliary tract cancers, 1037 biliary stones, and 959 controls), metabolic syndrome was defined as the presence of any three of the five components, including high waist circumference, high triglycerides, low high-density lipoprotein cholesterol (HDL), high blood pressure, and diabetes. Insulin resistance and ß-cell function were assessed, using homeostasis assessment models. RESULTS: Metabolic syndrome was significantly associated with gallbladder cancer (odds ratio (OR)=2.75, 95% confidence interval (95% CI)=1.82-4.15) and biliary stones (OR=1.64, 95% CI=1.24-2.16), with a significant dose effect with increasing number of metabolic syndrome components (P trend <0.0001). The observed association persisted among subjects without a history of diabetes. The association between insulin resistance and gallbladder cancer was borderline (P trend=0.06). There was a significant inverse association between ß-cell function and gallbladder cancer risk (P trend <0.001). CONCLUSION: Our findings suggest that metabolic syndrome and insulin resistance have a role in the aetiology of biliary tract cancers and biliary stones, and if confirmed, they imply that lifestyle control of these factors may lower the risk of biliary stones and biliary tract cancer.

Diabetes in relation to biliary tract cancer and stones: a population-based study in Shanghai, China.

BACKGROUND: Biliary tract cancers are rare but fatal malignancies. Diabetes has been related to biliary stones, but its association with biliary tract cancers is less conclusive. METHODS: In a population-based case-control study of 627 cancers, 1037 stones, and 959 controls in Shanghai, China, we examined the association between diabetes and the risks of biliary tract cancer and stones, as well as the effect of potential mediating factors, including serum lipids and biliary stones (for cancer), contributing to the causal pathway from diabetes to biliary diseases. RESULTS: Independent of body mass index (BMI), diabetes was significantly associated with gallbladder cancer and biliary stones ((odds ratio (OR) (95% confidence interval)=2.6 (1.5-4.7) and 2.0 (1.2-3.3), respectively). Biliary stones and low serum levels of high-density lipoprotein (HDL) were significant mediators of the diabetes effect on gallbladder cancer risk, accounting for 60 and 17% of the diabetes effect, respectively. High-density lipoprotein was also a significant mediator of the diabetes effect on biliary stones, accounting for 18% of the diabetes effect. CONCLUSIONS: Independent of BMI, diabetes is a risk factor for gallbladder cancer, but its effect is mediated in part by biliary stones and serum HDL levels, suggesting that gallbladder cancer risk may be reduced by controlling diabetes, stones, and HDL levels.

Reproductive factors and risks of biliary tract cancers and stones: a population-based study in Shanghai, China.

BACKGROUND: Parity has been linked to gallbladder cancer and gallstones, but the effects of other reproductive factors are less clear. METHODS: We examined 361 incident biliary tract cancer cases, 647 biliary stone cases, and 586 healthy women in a population-based study in Shanghai. RESULTS: The effects of parity (odds ratios, OR(> or =3 vs 1 child)=2.0, 95% confidence interval (CI) 0.7-5.1), younger age at first birth (OR(per 1-year decrease)=1.2, 95% CI 0.99-1.6), and older age at menarche (OR(per 1-year increase)=1.4, 95% CI 1.1-1.8) on gallbladder cancer risk were more pronounced among women with stones, but the interactions were not significant. CONCLUSION: Our results provide support for high parity, younger age at first birth, and late age at menarche in the development of gallbladder cancer, particularly among women with biliary stones.

Body size and the risk of biliary tract cancer: a population-based study in China.

Though obesity is an established risk factor for gall bladder cancer, its role in cancers of the extrahepatic bile ducts and ampulla of Vater is less clear, as also is the role of abdominal obesity. In a population-based case-control study of biliary tract cancer in Shanghai, China, odds ratios (ORs) and 95% confidence intervals (CIs) were calculated for biliary tract cancer in relation to anthropometric measures, including body mass index (BMI) at various ages and waist-to-hip ratio (WHR), adjusting for age, sex, and education. The study included 627 patients with biliary tract cancer (368 gall bladder, 191 bile duct, 68 ampulla of Vater) and 959 healthy subjects randomly selected from the population. A higher BMI at all ages, including early adulthood (ages 20-29 years), and a greater WHR were associated with an increased risk of gall bladder cancer. A high usual adult BMI (>or=25) was associated with a 1.6-fold risk of gall bladder cancer (95% CI 1.2-2.1, P for trend <0.001). Among subjects without gallstones, BMI was also positively associated with gall bladder cancer risk. Regardless of BMI levels, increasing WHR was associated with an excess risk of gall bladder cancer risk, with those having a high BMI (>or=25) and a high WHR (>0.90) having the highest risk of gall bladder cancer (OR=12.6, 95% CI 4.8-33.2), relative to those with a low BMI and WHR. We found no clear risk patterns for cancers of the bile duct and ampulla of Vater. These results suggest that both overall and abdominal obesity, including obesity in early adulthood, are associated with an increased risk of gall bladder cancer. The increasing prevalence of obesity and cholesterol stones in Shanghai seems at least partly responsible for the rising incidence of gall bladder cancer in Shanghai.

Total knee arthroplasty for severe haemophilic arthropathy: long-term experience in Taiwan.

A number of articles have investigated the outcomes of total knee arthroplasty (TKA) and causes of prosthetic failure in patients with haemophilic arthropathy. The aims of this retrospective study were to evaluate the clinical and functional outcomes of TKA and causes of prosthetic failure in patients with haemophilic arthropathy. A consecutive series of 35 TKA in 26 patients with haemophilic arthropathy were performed between November 1985 and October 2006 by one experienced surgeon. The mean age at index operation was 34.2 years old (range: 23.4-47 years) and the mean follow-up duration was 82.2 months (range: 12-218 months). Clinical assessment included range of flexion, range of extension and total range of motion (ROM). Functional evaluation comprised pain score and functional score by Dr. Insall's Knee Society Clinical Rating System. The average preoperative ROM was 63.2 degrees with flexion contracture 15 degrees , whereas the average postoperative ROM was 79.8 degrees with flexion contracture 5.5 degrees . Improvement of range of flexion was 7.1 degrees (P = 0.16); improvement of range of extension was 9.5 degrees (P < 0.01). Average increase of total ROM was 16.6 degrees (P = 0.02). Pain score by Knee Society was 7.1 points preoperatively and 48 points postoperatively (P < 0.01); functional score by Knee Society was 42 points preoperatively and 77.1 points postoperatively (P < 0.01). Three patients received manipulations because of an inadequate ROM. Three infection episodes were treated with debridement and one of them received arthrodesis after removal of prosthesis. Two patients received revision TKA. One of them was because of loosening of femoral component. The other one received revision TKA because of insert wear. Though improvement in range of flexion is insignificant in haemophilic arthropathy of knee after TKA, it showed significant increase in total ROM after operation, especially in improvement of flexion contracture. It also showed great pain relief and significant functional gain. Under the circumstance of acceptable infection rate and complication, TKA is an effective method to achieve pain relief and gain better function in patients with haemophilic arthropathy of knee. The data of this study confirm those previously published by many authors.

The spectrum of the factor 8 (F8) defects in Taiwanese patients with haemophilia A.

Haemophilia A (HA) is an X-linked recessive bleeding disorder caused by various types of pathological defects in the factor VIII gene (F8), which encodes coagulation factor VIII (FVIII). To date, several studies on the spectra of F8 defects have been performed in Western populations, but similar studies in Asian races are scarce. Here, we report the distribution of the mutations within the F8 gene in 31 Taiwanese unrelated HA patients (19 severe and 10 moderate/mild males and two severe females). Of these, 12 (38.7%) and one (3.2%) severe males were genotyped with the recurrent IVS22 and IVS1 inversion, respectively, similar to that in general populations (IVS22: 40-50%; IVS1: 2-5%). The F8 defects in the remaining 18 inversion-negative patients cover a wide spectrum, in which 17 different mutations were identified (10 missense and three nonsense mutations, and two small and two large deletions). Eleven of these mutations are novel: seven caused missense substitutions and four resulted in truncated proteins. To assess the putative pathogenetic impacts of the newly amino acid substitutions, computer analyses were performed based on molecular 3D modelling. The degree of conservation in cross-species FVIIIs and the position in known functional FVIII regions were studied. The novel missense mutations found in our series all occurred at evolutionary conserved residues that may carry a functional importance in our analyses. The results of this study add the short list of Taiwanese/Chinese F8 mutations, and will enhance our understanding of the molecular basis of FVIII function and the mechanism underlying HA.

Beta-catenin mutations in biliary tract cancers: a population-based study in China.

beta-Catenin is an ubiquitously expressed cytoplasmic protein that has a crucial role in both cadherin-mediated cell-cell adhesion and as a downstream signaling molecule in the wingless/Wnt pathway. Activating mutations in exon 3 of the beta-catenin gene, at the phosphorylation sites for ubiquitination and degradation of beta-catenin, are present in a variety of cancers. Because alterations of the adenomatous polyposis coli (APC) gene are present in biliary tract cancers and the APC protein modulates levels of beta-catenin, we evaluated the role of beta-catenin in biliary tract cancer by sequencing the third exon of the beta-catenin gene among 107 biliary tract cancers and 7 gallbladder adenomas from a population-based study in CHINA: Point mutations of serine or threonine phosphorylation sites in exon 3 of beta-catenin were present in 8 of 107 (7.5%) biliary tract cancers and 4 of 7 (57.1%) gallbladder adenomas. Mutations of beta-catenin were more frequent in ampullary and gallbladder carcinomas than in bile duct carcinomas (P = 0.04) and in papillary adenocarcinomas than other histological types of carcinomas (P = 0.02). These results suggest that the molecular pathways of biliary tract neoplasms vary by anatomical subsite and histological subtype.

Use of retinoic acids in the treatment of peripheral T-cell lymphoma: a pilot study.

PURPOSE: We have systemically analyzed, both in vitro and in vivo, the effect of 13-cis-retinoic acids (RA) on non-Hodgkin's lymphoma (NHL). METHODS: The in vitro growth-inhibitory effect of 13-cis-RA was examined in 11 (T cell, five; B cell, six) lymphoma cell lines by a tetrazolium colorimetric assay. A pilot clinical trial with oral 13-cis-RA 1 mg/kg/d was conducted in a selected group of 18 lymphoma patients, of whom 16 had failed to respond to at least one regimen of intensive chemotherapy. The in vitro and in vivo effects of 13-cis-RA were correlated with immunophenotypes, RA-induced changes of morphology, and patterns of DNA fragmentation of the lymphoma cells. RESULTS: Four of five T-lymphoma cell lines and none of six B-lymphoma cell lines were sensitive (concentration of 50% growth inhibition [IC50] < 1.5 microns) to 13-cis-RA (P = .015). In the clinical trial, five (two Ki-1, one angioinvasive type, one diffuse mixed cell, and one diffuse large cell) complete remissions and one (Ki1) partial remission were observed in 12 patients with peripheral T-cell lymphoma (PTCL), while none of six patients with B-cell lymphoma responded to 13-cis-RA. 13-cis-RA-induced cellular differentiation and apoptosis, as evidenced by the more mature morphology, characteristic nuclear condensation, and DNA ladder pattern signifying internucleosomal fragmentation, were demonstrated in the sensitive cell lines, as well as in the remitting lymphoma tissues. CONCLUSION: The 13-cis-RA appears to be active on lymphomas of T-lineage and their therapeutic indication may be extended to include some subtypes of PTCL. The mechanisms of action are related to differentiation and apoptosis of lymphoma cells. There appears to be no cross-resistance between 13-cis-RA and conventional chemotherapy.

Characterization of acute myeloid leukemia (AML) coexpressing lymphoid markers: different biologic features between T-cell antigen positive and B-cell antigen positive AML.

The clinical and biologic characteristics of acute myeloid leukemia (AML) with coexpression of lymphoid-associated antigens (Lym+ AML) were studied from 39 cases who represented 24% of 161 newly diagnosed de novo AML. Twenty-seven cases (16.8%) were positive for the expression of T-cell markers (T+ AML) and 12 (7.5%) for B-cell markers (B+ AML). Chromosomal abnormalities t(9;22)(q34;q11) and t/del(11)(q23), which were considered to be associated with acute leukemia coexpressing markers of more than one cell lineage, were detected in five and in four patients, respectively. There was no prognostic significance of B-cell or T-cell antigen expression in AML. Of 12 T+ AML cases in which cells were available for gene analysis, all showed germline configuration of immunoglobulin heavy chain and T-cell receptor beta chain genes, while seven of nine B+ AML showed rearrangements of either or both of the genes. Double labeling of the cells with myeloperoxidase and lymphoid markers demonstrated that individual blasts in all the five T+ AML tested were simultaneously expressing myeloperoxidase activity and CD7; however, most blasts in the three B+ AML studied expressed either myeloperoxidase activity or CD10, but not both. In eight of the nine T+ AML tested, the T-cell antigen-positive leukemic blasts were significantly decreased to less than 10%, after in vitro culture with the differentiation-inducing agent phorbol ester. B-cell markers remained positive (> or = 20%) on the cells in the two B+ AML who had the same study. These findings suggested that T+ AML and B+ AML might have different biologic features. Further studies on more patients are needed to clarify this point.

Extramedullary relapse after all-trans retinoic acid treatment in acute promyelocytic leukemia--the occurrence of retinoic acid syndrome is a risk factor.

All-trans retinoic acid (ATRA) is now a standard agent for remission induction of acute promyelocytic leukemia (APL). Recently, extramedullary relapse, which was a rare condition in APL patients after chemotherapy alone, was reported with an increased frequency after ATRA treatment. However, it is not yet clear whether ATRA truly increases the risk of extramedullary recurrence and what are the risk factors. In this study, three of 13 patients with recurrent APL after prior treatment of ATRA were found to have extramedullary involvement, compared with none in 11 recurrent patients previously treated with chemotherapy alone (estimated relative risk 2.100, 95% confidence interval 1.341-3.289). Furthermore, in the former group of patients, the development of retinoic acid (RA) syndrome during prior induction treatment was significantly associated with extramedullary involvement at relapse (three in five patients with RA syndrome vs none in eight without the syndrome, estimated relative risk 5.000, 95% confidence interval 1.448-17.271). In conclusion, ATRA may predispose APL patients to extramedullary involvement at relapse and the occurrence of RA syndrome is a risk factor for it. Further studies are needed to confirm these findings. It also remains to be clarified whether treatment modification is necessary in patients who develop RA syndrome during ATRA treatment.

Characterization of Philadelphia-chromosome-positive acute leukemia by clinical, immunocytochemical, and gene analysis.

Philadelphia chromosome (Ph') was detected at presentation in 10 out of 110 patients with acute lymphoblastic leukemia (ALL) and five of 168 patients with acute myelogenous leukemia (AML). Two other ALL patients who had studies at relapse were also included in the analyses. One of the 12 Ph'-positive (Ph+) ALL patients had simultaneous expression of myeloid-associated antigen on the leukemic blasts, while all the five AML patients coexpressed markers of lymphoid cells. Double labeling of the cells with myeloperoxidase and CD10 on three Ph+ AML cases showed that most leukemic blasts expressed either myeloperoxidase activity or CD10 but not both. Cross-lineage gene rearrangements of T-cell receptor (TCR) beta-chain gene were detected in three of the eight Ph+ ALL patients tested. All the four Ph+ AML cases studied showed immunoglobulin heavy chain gene rearrangements, and three of them also had simultaneous rearrangements of TCR beta-chain gene. The results revealed that Ph+ acute leukemia in this study belonged either to ALL or mixed lineage leukemia, and none was pure AML. This finding is contrary to that of acute blast crisis of chronic myelogenous leukemia in which the majority of patients had myeloid transformation. Rearrangements of bcr were detected in four of the 10 Ph+ ALL and three of the four Ph+ AML patients tested. No significant difference was noted in the clinical or hematologic manifestations among Ph+ leukemia with or without bcr rearrangements.

Clinical and biological characteristics of acute promyelocytic leukemia in Taiwan: a high relapse rate in patients with high initial and peak white blood cell counts during all-trans retinoic acid treatment.

Acute promyelocytic leukemia (APL) patients treated with all-trans retinoic acid (ATRA) and chemotherapy have been shown to have better outcome than those treated with conventional chemotherapy alone. However, the biological characteristics of leukemic cells and their clinical implications in patients treated with ATRA have not been well established. In this study, the biological and clinical features of 30 APL patients were reported. The risk factors for relapse and for occurrence of retinoic acid (RA) syndrome, which might cause morbidity or mortality of patients after ATRA treatment, were also analyzed. All patients showed 15;17 translocation by cytogenetic and/or gene analysis. Patients in this study had higher white blood cell (WBC) counts and a higher incidence of additional abnormalities than those from other areas. The ratio of long (L) form to short (S) form PML-RAR alpha fusion transcript was 1.8:1, a value lower than that of Latino patients but higher than that of Italians. Leukemic cells from four patients showed coexpression of T cell-associated antigen CD2 which was highly correlated with S form fusion transcript. Nine (36%) of the 25 patients treated with ATRA developed RA syndrome; all but one were successfully controlled by corticosteroid. Complete remission (CR) rate was 84%. Patients with high WBC counts tended to develop RA syndrome and had increased risk of relapse. Isochromosome for the long arm of the derivative chromosome 17, ider(17q), as an additional chromosomal abnormality was also associated with poor outcome in this study. In conclusion, APL in this study showed some different biological characteristics compared with those reported in other areas. High WBC count was a risk factor for relapse and development of RA syndrome after ATRA treatment. The prognostic implication of the presence of ider(17q) needs further clarification.

Clinical and biological implications of partial tandem duplication of the MLL gene in acute myeloid leukemia without chromosomal abnormalities at 11q23.

The clinical and biological features of acute myeloid leukemia (AML) with 11q23/MLL translocations are well known, but the characteristics of AML with partial tandem duplication of the MLL gene have not been explored comprehensively. In this study, MLL duplication was analyzed, in 81 AML patients without chromosomal abnormalities at 11q23, using Southern blotting, genomic DNA polymerase chain reaction (PCR), reverse-transcription PCR and complementary DNA sequencing. Nine patients showed partial tandem duplication of the MLL gene, including eight (12%) of the 68 with normal karyotype. Seven patients showed fusion of exon 6/exon 2 (e6/e2), one, combination of differentially spliced transcripts e7/e2 and e6/e2, and the remaining one, combination of e8/e2 and e7/e2. Among the patients with normal karyotype, children aged 1 to 15 showed a trend to higher frequency of MLL duplication than other patients (2/5 or 40% vs 6/62 or 10%, P = 0.102). The patients with tandem duplication of the MLL gene had a significantly higher incidence of CD11b expression on leukemic cells than did those without in the subgroup of patients with normal karyotype (75% vs 28%, P = 0.017). There were no significant differences in the expression of lymphoid antigens or other myeloid antigens between the two groups of patients. In adults, the patients with MLL duplication had a shorter median survival time than those without (4.5 months vs 12 months, P = 0.036). In conclusion, partial tandem duplication of the MLL gene is associated with increased expression of CD11b on leukemic blasts and implicates poor prognosis in adult AML patients. The higher frequency of MLL duplication in children older than 1 year, than in other age groups, needs to be confirmed by further studies.

Characterization of acute myeloid leukemia with MLL rearrangements--no increase in the incidence of coexpression of lymphoid-associated antigens on leukemic blasts.

MLL gene rearrangements are associated with coexpression of myeloid- and lymphoid-associated antigens on leukemic blasts and a dismal outcome in acute lymphoblastic leukemia (ALL). Whether the same conditions can apply to acute myeloid leukemia (AML) is not quite clear. Rearrangements of the MLL gene were analyzed on 113 patients with newly diagnosed de novo AML in a single institution. Sixteen (14%) of them showed rearranged bands by Southern blot analysis, including three (50%) of six infants, three (14%) of 21 children between 1 and 15 years and 10 (12%) of 86 adults. MLL rearrangements were not only detected in M5 (four of 12 patients, 33%) and M4 (six of 31, 19%) subtypes but also in other non-M4-M5 AML (six of 70, 9%), including M1, M2 and M7, but not M3 subtype. Seven patients had chromosomal abnormalities involving 11q23, but nine did not. The latter comprised three (6%) of 48 patients with normal karyotype, one with t(8;21), none with t(15;17), inv(16) or t(9;22), and four (15%) of 27 with cytogenetic aberrations other than those specific structural abnormalities. In contrast to ALL, AML patients with MLL rearrangements did not tend to coexpress lymphoid- and myeloid-associated antigens simultaneously on leukemic blasts and have similar outcome as those without the gene rearrangements.

Minimally differentiated acute myeloid leukemia in Taiwan: predominantly occurs in children less than 3 years and adults between 51 and 70 years.

Acute myeloid leukemia (AML) with minimal differentiation was usually referred to as acute undifferentiated leukemia in the past. With the help of immunophenotyping, this subtype of leukemia was shown to express myeloid antigens on the blasts and was designated AML-M0 by FAB Cooperative Study Group in 1991. Among the 423 consecutive newly diagnosed de novo AML at our institution, 12 (2.8%) were of M0 subtype. The proportion of M0 in AML was higher in children than in adults (8.2% vs 1.7%). Four other M0 patients referred from outside hospitals for immunophenotyping were also included in this study. There were two peaks in age distribution of these 16 patients: less than 3 years and between 51 and 70 years, respectively. Organomegaly was more common in patients with AML-M0 than in those with other subtypes (56.3% vs 29.2%, P = 0.025). The former patients had higher incidences of CD7 and CD34 expression on the leukemic cells than the latter ones (50% vs 16.9%, P = 0.003 and 69.2% vs 37.9%, P = 0.019, respectively). The patients with AML-M0 showed more frequent clonal chromosomal abnormalities in the leukemic cells than other AML patients (83.3% vs 53.9%, P = 0.039); the same is also true for complex cytogenetic aberrations (50% vs 11. 4%, P = 0.004). Adults with AML-M0 showed a lower complete remission (CR) rate and significantly poorer survival than those with non M0-AML. However there was no significant difference in outcome between the two groups of pediatric patients. In conclusion, AML-M0 is a unique subtype of leukemia that has distinct age distribution and shows different clinical and biological characteristics from other AML. Adult patients have poor prognosis. Whether pediatric patients had better outcome than adults needs to be clarified in further studies.

Characterization of genetic defects of hemophilia B of Chinese origin.

To define the precise genetic defects of hemophilia B of Chinese origin, we have used the polymerase chain reaction (PCR) combined with direct sequencing to analyze the amplified DNA fragments containing the entire coding regions and their flanking introns of the factor IX gene from 6 affected individuals. Among these patients, two are siblings with normal factor IX antigen level (CRM+) yet reduced factor IX clotting activity (28%). Analysis of their factor IX genes revealed a G to A transition at nucleotide residue 10394, which causes substitution of an arginine for a glycine at amino acid residue 48. This is a novel mutation which resides in the first EGF-like domain of factor IX. Studies of two other hemophilia B patients with CRMr phenotypes (factor IX antigen level less than 35%, and clotting activity less than 1%), demonstrated a distinct mutation in each individual's factor IX gene. In one case, a guanine to adenine (residue 6365) transition results in replacement of arginine by glutamine at the -4 codon of the propeptide of factor IX. In the other, thymine at 6442 was mutated to cytosine which causes an arginine for cysteine substitution at residue 23. We have also characterized 2 discrete CRM- patients. Both exhibited an identical mutation at nucleotide residue 6460 which generates a translation termination codon (CGA to TGA) at the 29th amino acid. The mutation created a new NlaIII restriction enzyme site which could be used to identify this variant.

Genetic basis and carrier detection of hemophilia B of Chinese origin.

We have characterized the genetic defects of 17 hemophilia B patients of Chinese origin by means of the polymerase chain reaction (PCR) and direct sequencing. The single-strand conformation polymorphism (SSCP) was used as an initial screening method to analyze the entire coding region and the flanking introns of each individual's factor IX gene. The abnormal exons were subsequently amplified and the nucleotide sequence determined. Of the 17 patients studied, 16 had single point mutations and one had a gross gene deletion of exons VII and VIII of factor IX. Among these 16 factor IX variants with point mutations 13 were missense and two were nonsense mutations. The remaining one had a nucleotide deleted, resulting in frame shifting at amino acid residue 97. A total of ten novel mutations, including the one with gross gene deletion, are reported in this study which have not been described previously. Five of the remaining seven variants were missense mutations with novel amino acids substituted for residues 127, 132, 180, 207, and 215, respectively. Mutations containing different amino acid residues at those positions have been reported. The last two are variants that have already been described to contain mutations at amino acid residues 333 and 365, respectively. To evaluate the efficiency of SSCP analysis in assessing the mutated exons and to further confirm our results we sequenced the entire exons of all 17 factor IX genes. The mutations detected by SSCP method were indeed the only mutation identified in each factor IX variant.(ABSTRACT TRUNCATED AT 250 WORDS)

Factor V Arg306 --> Gly mutation is not associated with activated protein C resistance and is rare in Taiwanese Chinese.

Polymerase chain reaction amplification followed by BstOI enzyme digestion and DNA sequencing was employed to detect the mutation of factor V gene. The subjects consisted of 105 venous thrombophilic patients and 183 healthy controls. Only one patient was found to have factor V Arg306 --> Gly mutation, his elder son also had an identical mutation. None of the healthy subjects studied had Arg306 --> Thr mutation. The rare event of factor V Arg306 --> Gly mutation in patients and controls suggest that this mutation is not associated with increased risk of venous thrombosis. Conventional, modified and extended activated protein C (APC) resistance assays in this patient and his family members clearly showed that factor V Arg306 --> Gly mutation is not associated with APC resistance (APC sensitivity ratio <2). In conclusion, factor V Arg306 --> Gly mutation is rare in Taiwanese Chinese and not associated with APC resistance, it is possibly not a risk factor for venous thrombophilic thrombosis.