Biomedical subjects

D Martincic

Publications and source records attributed to D Martincic.

9 recordsLinked to original sources

PubMed2001

Defective binding of factor XI-N248 to activated human platelets.

Variants of factor XI containing Gln226 to Arg (Q226 to R) and Ser248 to Asn (S248 to N) substitutions were first identified in an African American family with a history of excessive bleeding. The substitutions have recently been identified in unrelated individuals, suggesting they are relatively common. Both amino acids are located in the third apple domain of factor XI, an area implicated in binding interactions with factor IX and activated platelets. Recombinant factor XI-R226 and factor XI-N248 were compared with wild-type factor XI in assays for factor IX activation or platelet binding. Factor XI-R226 activates factor IX with a Michaelis-Menten constant (K(m)) about 5-fold greater than wild-type protein. The catalytic efficiency of factor IX activation is similar to wild-type protein, however, due to an increase in the turnover number (k(cat)) for the reaction. Iodinated factor XI-N248 binds to activated platelets with a dissociation constant (K(d)) more than 5-fold higher than wild-type protein (55 nM and 10 nM, respectively). Activation of factor XI-N248 by thrombin in the presence of activated platelets is slower and does not progress to the same extent as activation of the wild-type protein under similar conditions. Factor XI-N248 activates factor IX normally in a purified protein system and has relatively normal activity in activated partial thromboplastin time (aPTT) assays. Factor XI-N248 is the first factor XI variant described with a clear functional difference compared with wild-type protein. Importantly, the defect in platelet binding would not be detected by routine clinical evaluation with an aPTT assay.

Amino Acid Substitution↗

PubMed2000

Conserved worldwide linkage disequilibrium in the human factor XI gene.

We have identified, in four diverse human populations, five common single-nucleotide polymorphisms (SNPs) in the coding region of the gene for the blood coagulation protease factor XI. Each SNP has an allele frequency >5% in at least one population. Three of the SNPs (C472T, A844G, and T1234C), spread out over approximately 10 kb of genomic DNA, are in marked linkage disequilibrium (LD) with one another (P < 10(-4)). Interestingly, haplotypes associated with the linked SNPs are conserved across all populations studied, despite significantly different allele frequencies between populations. The presence of such common, widely dispersed haplotypes could complicate the interpretation of LD studies and emphasizes the need for a better understanding of general patterns of LD to facilitate identification of genes for common disorders.

Factor XI↗

PubMed2000

Familial thrombophilia associated with fibrinogen paris V: Dusart syndrome.

We report on a family with a history of venous thromboembolism associated with fibrinogen Paris V (fibrinogen Aalpha-Arg554-->Cys). Ten members experienced thrombotic events, including 4 with fatal pulmonary emboli. Pulmonary embolism was the presenting feature in 4. Those with the mutation and a history of thrombosis had somewhat higher fibrinogen concentrations than those with the mutation and no thrombosis (294 +/- 70 mg/dL vs 217 +/- 37 mg/dL, respectively). The Paris V mutation consistently caused a prolongation of the reptilase time, and fibrin clots containing the abnormal fibrinogen were more translucent than normal clots. Given the early onset of symptoms and the initial presentation with pulmonary embolism in some family members, it was justifiable to offer prophylactic anticoagulation with warfarin to carriers of the mutation. Fibrinogen Paris V has now been reported in 4 apparently unrelated families, indicating that it is a relatively common cause of dysfibrinogenemia-associated thrombosis.

Adolescent↗

PubMed1999

Factor XI messenger RNA in human platelets.

The bleeding diathesis associated with congenital deficiency of factor XI (FXI) is variable and correlates poorly with standard coagulation assays. Platelets are reported to contain FXI activity that may substitute for the plasma protein. The presence of this platelet activity in some patients deficient in plasma FXI could partly explain the variable bleeding associated with the deficiency state. Polyclonal antibodies to plasma FXI recognize a 220 kD platelet membrane protein distinct in structure from plasma FXI. The messenger RNA (mRNA) coding for this protein has been postulated to be an alternatively spliced FXI message lacking the fifth exon found in the liver (wild type) message. We analyzed RNA from platelets, leukocytes, and bone marrow for FXI mRNA by reverse transcription polymerase chain reaction (RT-PCR) technology. Single FXI mRNA species were identified by RT-PCR in platelet and bone marrow RNA, but not leukocyte RNA, that are the same size as the message from liver RNA. Sequencing of PCR products confirmed that the FXI mRNA species in platelets is identical to the one in liver. Wild-type FXI mRNA was also identified in three leukemia cell lines with megakaryocyte features (MEG-01, HEL 92.1.7, and CHRF-288-11). The data show that platelets contain wild-type FXI mRNA. FXI protein, therefore, may be present in platelets and may be released during platelet activation. The data do not support the premise that the 220 kD platelet protein that cross-reacts with FXI antibodies is a product of an alternatively spliced mRNA from the FXI gene.

Alternative Splicing↗

PubMed1999

Detection of mutations by automated fluorescence/RNA-based dideoxy fingerprinting (ARddF)

Dideoxy fingerprinting (ddF) is a hybrid technique which combines aspects of single strand conformational polymorphism (SSCP) and dideoxy sequencing to detect the presence of single base changes in a defined fragment of nucleic acid. ddF is no more technically demanding than SSCP, yet it is more sensitive in detecting point mutations. We describe here the adaptation of conventional ddF to an automated sequencing system using fluorescent Cy5 labeled primers. We show that automated RNA-based ddF (ARddF) has several advantages over conventional radioisotope-based ddF, including: (1) analysis of larger nucleic acid fragments (up to 10(3) bp), due to the ability to continuously analyse and compile sequencing information; (2) greater reliability for distinguishing mutant sequences from wild type sequences (particularly when the mutation leads to gain or loss of a dideoxy termination segment); (3) the use of fluorescent labeled primers, making ARddF less hazardous than methods requiring radionucleotides. The use of ARddF in conjunction with new methods for isolating RNA from a [corrected] small number of cells facilitates mutational analysis of small tissue biopsies and other limited samples, and will allow more widespread application of mutational screening in the setting of clinical diagnostic laboratories.

Animals↗

PubMed1998

Identification of mutations and polymorphisms in the factor XI genes of an African American family by dideoxyfingerprinting.

Congenital deficiency of factor XI is a rare condition associated with a mild to moderate bleeding diathesis that is most commonly found in persons of Jewish ancestry. The disorder has been reported sporadically in a number of other ethnic groups, but rarely in the black population. We report on the genetic analysis of the factor XI genes of two African American patients: a 9-year-old boy (the propositus) with mild factor XI deficiency and his mother. Both individuals have lifelong histories of excessive bleeding. Dideoxyfingerprinting, a technique combining components of single-strand conformational polymorphism analysis and dideoxy-chain termination sequencing, was used in the analysis. Both patients were found to be heterozygous for a mutation changing serine 248 to asparagine [corrected], whereas the propositus was heterozygous for an additional mutation on the paternal allele changing glutamine 226 to arginine. Both mutations reside in the third apple domain of the factor XI heavy chain, an area that has been shown to contain binding sites for factor IX, platelets, and glycosaminoglycans. Previously reported mutations in the factor XI gene seem to cause deficiency primarily by reducing protein expression. Because both alleles in the propositus contain amino acid substitutions, the significant amount of circulating factor XI in his plasma must be comprised entirely of abnormal molecules. Factor XI circulates as a homodimer, and the presence of mutations in both alleles of the factor XI gene suggests that his bleeding disorder is caused in part by the effect of the two abnormal gene products forming dimers in different combinations. Three neutral (not associated with amino acid changes) DNA polymorphisms were also identified in the two subjects: a C to T change at nucleotide 472 in exon 5, A to G at nucleotide 844 in exon 8, and T to C at nucleotide 1234 in exon 11. Analysis of a random sample of normal volunteers showed that these polymorphisms are relatively common, with allele frequencies of 7.4%, 19%, and 18%, respectively. This suggests that there is considerable genetic heterogeneity in the factor XI gene.

Adult↗

PubMed1997

Folate deficiency delays the onset but increases the incidence of leukemia in Friend virus-infected mice.

Clinical studies have indicated that folate deficiency may enhance the development of various malignancies. In animal studies that examined the effect of folate deficiency on malignancies, conflicting results have been reported. In some studies, folate deficiency increased the development and growth of malignant tumors; in others, it decreased the development and growth of malignancies. We examined the effect of transient folate deficiency on the development of leukemia in mice infected with the anemia-inducing strain of Friend leukemia virus. Friend virus disease can be considered as a model for human acute leukemias that are preceded by a preleukemic period. These include leukemias that develop in patients who received previous chemotherapy and/or radiation therapy, as well as patients with chronic granulocytic leukemia or myelodysplasia. Folate deficiency around the time of Friend virus-infection delayed the onset but increased the incidence of leukemia. The rates of rearrangement of the Spi-1 (PU.1 ) oncogene by provirus integration and alteration of the p53 tumor-suppressor gene were the same in leukemia cell lines derived from folate-deficient mice as they were in cell lines from control mice. These results indicate that folate deficiency did not exert its enhancement of leukemogenesis through changes in either Spi-1 or p53, even though these two genes have been found to be the most frequently altered ones in Friend virus-induced leukemias. Our results suggest that folate deficiency may enhance the development of acute leukemia in patients who are at high risk for this disease.

Animals↗

PubMed1997

Transforming growth factor beta as a potential tumor progression factor among hyperdiploid glioblastoma cultures: evidence for the role of platelet-derived growth factor.

Among early-passage, near-diploid gliomas in vitro, transforming growth factor type beta (TGF beta) has been previously shown to be an autocrine growth inhibitor. In contrast, hyperdiploid (> or = 57 chromosomes/metaphase) glioblastoma multiforme (HD-GM) cultures were autocrinely stimulated by the TGF beta. The mechanism of this 'conversion' from autocrine inhibitor to mitogen is not understood; previous studies have suggested that platelet-derived growth factor (PDGF) might be modulated by TGF beta. The similar expression of TGF beta types 1-3, PDGF-AA; -BB, as well as the PDGF receptor alpha and beta subunits (a/beta PDGFR) between biopsies of the HD-GM and near-diploid, TGF beta-inhibited glioblastomas (GM) by immunohistochemistry did not explain the discrepancy in their regulatory responses. Flow cytometry demonstrated that TGF beta's mitogenic effect was selective for the aneuploid subpopulations of two of three selected HD-GM cultures, while the diploid cells were inhibited. Among the HD-GM, TGF beta 1 induced the RNA of PDGF-A, c-sis and TGF beta 1. The amount of PDGF-AA secreted following TGF beta treatment was sufficient to stimulate the proliferation of a HD-GM culture. Antibodies against PDGF-AA, -BB, -AB, alpha PDGFR and/or beta PDGFR subunits effectively neutralized TGF beta's induction of DNA synthesis among the HD-GM cell lines, indicating that PDGF served as the principal mediator of TGF beta's growth stimulatory effect. By comparison, TGF beta induced only the RNA of PDGF-A and TGF beta 1 among the near-diploid GM, c-sis was not expressed at all. However, the amount of PDGF-A which was secreted in response to TGF beta 1 was insufficient to prevent TGF beta's arrest of the near-diploid cultures in G1 phase. Thus, the emergence of hyperdiploidy was associated with qualitative and quantitative differences in TGF beta's modulation of PDGF-A and c-sis, which provided a mechanism by which the aneuploid glioma cells might achieve 'clonal dominance'. We hypothesize that TGF beta may serve as an autocrine promoter of GM progression by providing a selective advantage to the hyperdiploid subpopulation through the loss of a tumor suppressor gene which mediates TGF beta's inhibitory effect.

Aneuploidy↗

PubMed1996

Improved detection of p53 point mutations by dideoxyfingerprinting (ddF).

Two screening techniques for identifying point mutations (single-strand conformational polymorphism (SSCP) and dideoxyfingerprinting (ddF)) were compared to sequencing to determine their efficiency in detecting mutations in exons 5-8 of the p53 tumor suppressor gene. Twelve human glioblastoma cell lines were studied by each of the three methods. Ten mutations were identified by sequencing; of these, 10/10 were detected by ddF, while SSCP detected 6/10 true mutations and falsely identified two presumed mutations not confirmed by sequencing. We examined the impact of parameters which influence DNA conformation (gel temperature, gel composition, and PCR product size) on the ability of SSCP and ddF to detect mutations. The sensitivity of SSCP varied with both gel temperature and the size of the PCR product; in contrast, ddF was not influenced by either gel temperature or product length (up to 460 nucleotides). We conclude that the increased sensitivity of ddF, together with its greater ease of application due to the lack of need for optimization, provides significant advantages over SSCP in screening DNA sequences for the presence of point mutations. Our results also suggest that the incidence of p53 mutations may be underestimated in studies of human cancers which utilize SSCP as the method of mutational screening.

DNA↗