CK Domain Research Articles

Diagnosis of TPACK in Elementary School Teachers: A Case Study in the Colombian Caribbean

Numerous studies support that the development of digital teaching competence is essential in 21st century schools. This paper examines Technological Pedagogical Content Knowledge (TPACK) to gain a deeper understanding of ICT integration in teaching. By assessing TPACK, we uncover opportunities to enhance teacher competencies and, consequently, improve student learning. This research evaluated the initial TPACK of primary school teachers from a public school in the Colombian Caribbean. Eight teachers participated in a professional development program based on the Lesson Study (LS) methodology. Adopting an interpretive qualitative approach and a case study for the operational analysis of LS, the findings indicate that teachers, in self-reports and performance-based assessments, highlight high competence in the PK, CK, and PCK domains. This demonstrates their ability to select and adapt effective teaching strategies. They excel in guiding learning and understanding academic content, showcasing a remarkable capacity to adapt to the diverse socioeconomic realities of their students. However, these findings also highlight areas for improvement in developing the technological components of TPACK, specifically TK, TPK, TCK, TPCK, and XK.

A Mixed Methods Study to Address the Integration of TPACK in Elementary School Students' Learning Process

The Technological Pedagogical and Content Knowledge (TPACK) framework offers guidance for teachers seeking to address technology integration in classroom learning. This study aims to assess the TPACK proficiency levels among elementary school teachers. This study involved teachers from SDN Sumbersari 03 in Malang City as participants using a mixed-methods approach with a sequential explanatory design. Quantitative data were initially gathered through self-report measures, followed by analysis utilizing descriptive and inferential statistics. Subsequently, qualitative data were collected through interviews and observations, with analysis conducted using the interactive model proposed by Miles and Huberman. The results of this study showed that there was no difference in the TPACK perceptions of primary school teachers based on gender (p-value > 0.05). However, there is an influence between age and tenure on teachers' TPACK ability (p-value < 0.05). Overall, the teachers have a good mastery of TPACK. However, there are some areas that need to be improved, especially in terms of technology mastery (TK, TCK, and TPACK). The highest mean score in the CK and PK domains was 4.875 and the lowest score in the TPACK domain was 4.55. The findings also indicate no significant difference in TPACK perceptions among elementary school teachers based on gender. However, age and tenure were found to influence teachers' TPACK abilities. Overall, teachers demonstrated a good level of mastery in TPACK. Nonetheless, specific areas, particularly technology mastery (TK, TCK, and TPACK), require improvement.

PB0855 New Mutation in CK Domain of VWF Gene in Patient with Type 2N von Willebrand Disease

PB0855 New Mutation in CK Domain of VWF Gene in Patient with Type 2N von Willebrand Disease

Cross-Sectional Survey: The Influence of Various Teacher Backgrounds on West Kalimantan Chemistry TPACK Teachers

This study aims to examine teachers' perceptions of their Technological Pedagogical and Content Knowledge (TPACK) about age, gender, length of teaching, work location, teacher status, teacher education, and certification programs. This study used a cross-sectional survey method used in non-experimental quantitative research. The population in this study were all chemistry teachers in West Kalimantan using a purposive sampling technique obtain 70 to respondents from 14 districts. The survey consisted of 28 statement items and distributed questionnaires via Google form to chemistry teachers. Chemistry teachers rated themselves on a 5-point scale. All domains of the TPACK framework demonstrated valid and reliable instruments on Pearson's correlation values and Cronbach's Alpha Kruskal Wallis non-parametric test, Pearson's correlation was performed to analyze the data. Significant differences can be seen in the CK domain when viewed from gender differences with a p for CK of 0.044, so there is a significant difference between the gender of women (Mean = 4.08 SD = 0.416) and men (Mean = 4.32 SD = 0.470). Location of work affects the teacher's TCK and TPACK because there is a significant difference when viewed from the p namely the TCK and TPACK of 0.036 and 0.025. Perceptions of teachers' TPACK abilities were greater for teachers in cities than in villages in both the TCK domain (Mean = 3.58; 3.82 SD = 0.430; 0.466) and TPACK (Mean = 3.63; 3.91 SD = 0.483; 0.554).

Modular Cloning of MUC1 Recombinant Antibodies by Assembly of Synthetic Domain Genes

Antibody immunotherapy has emerged as a constructive way to fight cancer. New immunotherapies require the identification of a tumor‐specific antigen. The protein mucin 1 (MUC1) has long been recognized as an important specific antigen and a potential immunotherapy target. In healthy human tissues, MUC1 is responsible for protecting the epithelial lining. However, in cancer cells, MUC1 displays truncated glycosylation, revealing tumor‐specific epitopes. Recombinant antibodies are preferred for therapeutic development, and in some cases, it is desirable to produce different antibody formats, including intact IgG or Fab fragments. Using the MUC1 specific antibody 4H5 as a model, we have developed a modular cloning approach that can rapidly produce different antibody modalities to facilitate the rapid production of both IgG and Fab antibody formats. The 4H5 variable domains were PCR amplified to contain 20 base‐pair (bp) overlaps with the pcDNA3.1 vector on the 5’ end, and 20 bp overlaps to either the human CH1 or Ck domains on the 3’end of the genes. To produce the Fab, a His‐tag and a 20 base‐pair (bp) overlap to the vector was added to the 3’ end of a human CH1 domain gene sequence. For IgG production, human CH1‐CH2‐CH3 domains were PCR amplified, and the same vector overlap sequence was added to the 3’ end of the sequence. Finally, a 20 bp overlap to the vector was added to the 3’ end of a human Ck domain. The PCR products were purified, and the constructs were assembled using the NEBuilder®HiFi DNA Assembly Cloning Kit. DNA sequencing confirmed the successful assembly of the coding sequence, and the 4H5 Fab fragments and IgG were produced by transient transfection in CHO cells. The antibodies (Fab and IgG) were purified by affinity chromatography and binding to recombinant MUC1 antigens confirmed by ELISA. The cloning method presented here simultaneously produces multiple antibody formats. This approach will permit rapid assembly and screening of different antibody formats and isotypes for therapeutic antibody discovery and analysis.

Traces and extensions of generalized smoothness Morrey spaces on domains

We study traces on the boundary of generalized smoothness Morrey spaces on Ck domains Ω. These spaces are equipped with three parameters s,p,q and a function parameter φ. Our results remain valid for the usual Besov–Morrey spaces Nu,p,qs(Ω), Triebel–Lizorkin–Morrey spaces Eu,p,qs(Ω), and Triebel–Lizorkin type spaces Fp,qs,τ(Ω), which are all included in our scales as special cases. Moreover, to complete our investigation, we also study traces of Besov-type spaces Bp,qs,τ(Ω), which are not covered by our approach and give some applications in terms of a priori estimates generalizing results from El Baraka (2005).

Distribution of Pathogenic Variants in Von Willebrand Disease Type 2A in the American Population: Results from a National Clinical Reference Laboratory

Introductionvon Willebrand disease (VWD) is a common inherited bleeding disorder with a reported incidence ranging from 0.01% to 1%. VWD is classified as quantitative (types 1 and 3) and qualitative (type 2) defects in von Willebrand factor (VWF). Classical VWD type 2A, the most common qualitative defect of VWD, is characterized by decreased VWF-dependent platelet adhesion and a selective deficiency of high molecular weight VWF multimers. The majority of type 2A VWD is inherited in an autosomal dominant fashion. This subtype is caused by missense variants acting through a variety of mechanisms including defects in multimer assembly or an intrinsically increased sensitivity to cleavage by ADAMTS-13. The majority of pathogenic mutations are located in exon 28, predominantly affecting the A2 domain. Missense mutations have also been reported in the D3 and A1 domains (exons 22 and 25-28). Less common missense mutations include those responsible for dimerization (CK domain, exon 52) and multimerization defects (D2 domain, exons 11-17). Our objective was to determine the genomic distribution of mutations underlying type 2A VWD in patients referred to a USA-based clinical reference laboratory.MethodsIn a clinical reference laboratory, BloodCenter of Wisconsin, VWF gene sequence analysis results from January 2009 to July 2018 were reviewed. Molecular testing was performed by polymerase chain reaction (PCR) amplification and bi-directional Sanger sequencing or capture-based enrichment and next-generation sequencing (NGS). Complete coding region and splice junction of VWF gene was compared to the reference sequence (NM_00552.3). Variant interpretation was performed according to American College of Medical Genetics (ACMG) standards and guidelines by utilizing genome databases, population frequency data, variant specific literature search and predictive computational tools. Variant interpretation was reviewed by a team comprising of genetic counselors, molecular experts and experts in coagulation disorders correlating these findings with the patient's clinical phenotype provided.ResultsPathogenic variants were identified in 46 cases received. Of these, 56.5 % (26 of 46) were in exon 28 of the VWF gene.(Figure 1) 21.7% of mutations (10 out of 46) were identified in exon 26, 4.3% were identified in exon 25 (2 of 46) and 6.5% (3 out of 46) were identified in exon 52 of VWF gene. The remaining 10.7% were identified in exons 6,11,12,22 and 27 (1 each). The distribution of these mutations according to VWF domains was also studied. 39% of these mutations were found in the A2 domain, 30.4% were identified in the D3 domain, and 13% were found in the A1 domain. A small number of these mutations were identified in the D1 domain (1/46), D2 domain (2/46) and CK domain (2/46). Phenotypic information was available for most patients evaluated.ConclusionsIn a predominantly Caucasian population with others of mixed ethnicities, the distribution of VWD type 2A mutations was studied in patients evaluated in a clinical reference laboratory. Most mutations were identified in VWF Exon 28 and in the A2 domain. Next generation sequencing platform enables sequencing of large amounts of DNA in parallel thereby allowing all VWF exons to be evaluated at one time. However, in smaller laboratories, without access to a NGS platform, initial evaluation of the VWF Exon 28 for evaluation of VWD type 2A variants is a cost effective measure. Subsequently, sequencing of other less implicated exons may be performed utilizing a reflexive algorithm. Genetic testing can be very useful in diagnosis of type 2 variants, where mutations in specific regions correspond to the defect in VWF function. [Display omitted] DisclosuresFriedman:Shire: Membership on an entity's Board of Directors or advisory committees; CSL Behring: Membership on an entity's Board of Directors or advisory committees.

Domain Distribution of Type 1 VWD Sequence Variants and Impact on Clinical and Laboratory Phenotype in the Zimmerman Program

Type 1 von Willebrand disease (VWD) is a common inherited bleeding disorder, characterized by a quantitative deficiency of von Willebrand factor (VWF). The molecular basis continues to merit exploration, especially since the genetic cause has not been determined in 30-40% of cases in previous studies. As part of the Zimmerman Program for the Molecular and Biology of VWD (ZPMCB-VWD) study, we sought to identify the sequence variants (SV) within the VWF gene that result in type 1 VWD and correlate these to clinical laboratory phenotype and clinical severity and determine the frequency of type 1 subjects who do not have a gene defect. We also wanted to examine the types of sequence variants that cause type 1, type 1-Severe (1S), type 1C and low VWF (LVWF), and determine the influence of SV location on VWF levels and risk of bleeding. 310 type 1 Index Cases (IC) enrolled in the ZPMCB-VWD were analyzed, including 69 type 1 (VWF:Ag<30), 9 type 1S (VWF:Ag<5), 57 type 1C (VWF:Ag<30; VWFpp/VWF:Ag>3), and 175 LVWF (VWF:Ag or VWF:RCo 30-50). Bleeding symptoms were quantified using the ISTH bleeding score (ISTH BS). Clinical VWF testing was done in a central laboratory at BloodCenter of Wisconsin and included FVIII, VWF:Ag, VWF:RCo, VWFpp, and VWF:CB. Full-length VWF sequencing was performed, including 5' and 3' regions and intron/exon boundaries. SV found in >1% of the healthy control population were excluded from this analysis. Mutation negative cases were further investigated by comparative genomic hybridization (CGH) to identify any large deletions or duplications in the VWF gene. SV or large deletions were identified in 62% (n=192) of this cohort with quantitative VWF deficiency, whereas 38% (n=118) had no variants found. Of those with no sequence variant identified, 10% were type 1, 5% type 1C and 85% LVWF. There were 142 SV found in this cohort. 75% of the subjects had missense mutations, 8% small insertions/deletions, 9% splice site mutations, 3% nonsense mutations and 1% large deletions. The median number of SV found in individuals was 1, however 23% had more than 1 SV present (range 1 to 4). Mean VWF:Ag (IU/dl) was examined to determine the influence of SV location on levels and median ISTH BS was assessed as a measure of bleeding severity by domain: Table.VWF DomainD1D2D'D3A1A2A3D4C1-C6CKNone# Subjects24343460251324304118VWF:Ag (IU/dl)18211614311221253839ISTH BS5665495515 Type 1S subjects had SV that were enriched in D1, D2 and A3 domains. 52% of type 1C variants were found in the A1 domain followed by 19% in D'D3 and 17% in D4. Both type 1 and LVWF subjects had SV that were evenly distributed throughout the gene, but LVWF had the majority of SV found in the A2, D2 and C1-C6 domains. Type 1S subjects showed a difference in mutation type compared to rest of the type 1 cohort with 31% intronic/splice site mutations, 19% nonsense mutations, 19% insertions/deletions and only 25% missense mutations. In contrast, missense mutations accounted for 68% of the mutations in type 1, 91% in type 1C and 80% in LVWF. The presence of predominantly truncating or loss of function variants in type 1S correlates with lower VWF levels and increased bleeding. In conclusion, we found that 62% of our type 1 cohort did have potentially causative SVs identified, with the majority caused by missense mutations (75%). Type 1C and type 1S tend to have SV isolated in certain domains whereas SV in type 1 and LVWF tend to be spread throughout the gene. SVs in the A3 domain demonstrated the lowest VWF:Ag (12 IU/dl) and highest ISTH BS (9) while changes in the CK domain had the highest VWF:Ag (38 IU/dl) and lowest ISTH BS (1). SVs or large deletions were not found in 38% of this cohort, indicating that other molecular factors either intronic or outside of the VWF gene may be the cause of low VWF and bleeding in these subjects. Disclosures Friedman: CSL Behring: Consultancy; Instrumentation Laboratories: Consultancy; Novo Nordisk: Consultancy; Alexion: Speakers Bureau. Gill:Baxalta, Bayer, and CSL-Behring: Membership on an entity's Board of Directors or advisory committees.

Heterogeneity Of Type 3 Von Willebrand Disease (VWD): Evidence From Patient-Derived Blood Outgrowth Endothelial Cells (BOEC)

BackgroundType 3 von Willebrand Disease (VWD) is the rarest and most severe form of the disease. Patients exhibit plasma von Willebrand factor (VWF) levels of<5% for both VWF:Ag and VWF:RCo and have low FVIII levels (<10%). Patient-derived blood outgrowth endothelial cells (BOEC) provide a useful cellular model for studying VWD instead of using heterologous cell systems, which do not completely mimic the native environment. Until recently, we believed the lack of endogenous VWF in patients with type 3 VWD might pose as a challenge for isolating and culturing type 3 VWD BOEC. The objectives of the current study were to isolate BOEC from type 3 VWD patients with different genotypes and to compare the cellular presence, if any, and localization of VWF in the patient-derived BOEC using confocal immunofluorescence microscopy. Patients and MethodsPeripheral blood samples were obtained from seven type 3 VWD index cases (IC) (from six different families) in order to isolate and culture BOEC. These patients were originally enrolled in the Canadian type 3 VWD study as IC or siblings of IC. Research Ethics Board approval was obtained, as well as informed consent from all participants. Endothelial cell phenotype of isolated cells was confirmed using flow cytometry for cell surface markers; positive for CD31, CD144 and/or CD146, and negative for CD14 and CD45. Confocal immunofluorescence microscopy was used to observe intracellular VWF. ResultsWe were able to successfully isolate and culture BOEC from seven type 3 VWD patients with different genotypes. All cells were confirmed as endothelial cells as per the markers listed in the methods. Table 1 shows the patient characteristics, and corresponding VWF phenotype and genotype.Table 1Type 3 VWD Index Case Phenotype and GenotypePatient IDGender/age (M/F year)Bleeding ScoreVWF:Ag (IU/ml)VWF:RCo (IU/ml)FVIII:C (IU/ml)Nucleotide Change, HGVSAmino Acid Change, HGVSExonGenotypeT001M/42190.010.020.02c.221-977_532+7059delp.Asp75_Gly178del4-5HomozygousT050F/21240.010.000.01c.876delC, c.1255C>Tp.Ser293fs, p.Gln419*8/11Compound heterozygousT076M/17230.010.030.01c.1897T>Cp.Cys633Arg15HomozygousT076-SF/1190.010.030.01c.1897T>Cp.Cys633Arg15HomozygousT105F/62180.020.000.03c.8419_8422dupTCCCp.Pro2808Leufs*2452HomozygousT151M/27290.020.040.01c.3939G>A, c.5842+1 G>Cp.Trp1313*, -28/34Compound heterozygousT154M/16240.010.030.01c. 1657dupT, c.8419_8422dupTCCCp.Trp553Leufs*97, p.Pro2808Leufs*2414/52Compound heterozygousDespite a virtual lack of VWF in the plasma of the seven type 3 VWD patients, confocal immunofluorescence microscopy studies showed different patterns of intracellular VWF. Qualitative and/or quantitative defects in Weibel-Palade bodies (WPB) were observed. T001, T076, and T076-S, IC whom are homozygous for VWF propeptide mutations, show a complete lack of WPB formation, with only diffuse staining of VWF, indicative of retention in the endoplasmic reticulum (ER). This was confirmed by co-localization of the diffuse VWF with the ER marker calnexin. T105 and T154, who have in common a frameshift mutation in the CK domain of VWF showed similar VWF staining patterns with diffuse staining and a limited number of rounded WPBs. T151, a compound heterozygote for a nonsense mutation in exon 28 and a splice site mutation in intron 34, showed both diffuse VWF staining, as well as a number of rounded WPBs. The latter three cases indicate abnormalities in WPB biogenesis which would impair secretion of VWF. T050, who is compound heterozygous for two different VWF propeptide mutations, showed a complete absence of VWF, presenting a true VWF “null” endothelial cell. DiscussionWe have shown that BOEC can be successfully isolated from type 3 VWD patients with different VWF genotypes. As well, we have further confirmed the heterogeneity in type 3 VWD as seen in the variety of endothelial cellular phenotypes associated with the different VWF genotypes. Patient-derived BOEC are a useful cellular model for investigating the pathogenic nature of VWF mutations reflective of the native vascular environment. As well, they may be a useful tool to study the effect of novel treatments for VWD, leading the way for personalized treatment of VWD. Disclosures:James:CSL Behring: Honoraria, Research Funding; Octapharma: Honoraria, Research Funding; Baxter: Honoraria; Bayer: Honoraria.

Overcoming antibody expression and screening limitations by smart design: Applications to PSA immunoassay development

Improving the functional and structural properties of target proteins can often be a challenge for researchers. This paper highlights the importance of antibody construct on screening performance, and ultimately, the clone that is selected. We report the reformatting of phage-selected single chain antibody variable region fragments (scFvs) into single chain antibody fragments (scAbs) for improved screening and binding studies. The generation of a scAb, which had a fused human kappa light chain constant domain (Ck), was shown to significantly improve expression levels in Escherichia coli. Antibody expression levels were compared between the two antibody constructs (scFv and scAb) by ELISA and a 100-fold improvement was observed. The Ck domain in the expressed scAb also facilitated high throughput analysis by a Biacore capture assay approach. Individual functional scAbs were ranked on the basis of their remaining binding percentage after 5min dissociation. Selected antibodies were further characterised by kinetic analysis and a sandwich-based immunoassay developed. The scAb construct enhanced expression levels significantly, facilitating antibody screening and immunoassay development for prostate-specific antigen (PSA), a marker for prostate cancer.

A Class of Atomic Rings

Let R be a commutative ring with identity. A nonunit element a ∈ R is an atom if a = bc (b, c ∈ R) implies (a) = (b) or (a) = (c), and R is atomic if each nonunit of R is a finite product of atoms. A ring R is a (weak) CK ring if R is atomic and (each maximal ideal of) R contains only finitely many nonassociate atoms. We show that the following are equivalent: (1) R is a weak CK ring, (2) each (prime) ideal of R is a finite union of principal ideals, (3) R is atomic and each maximal ideal is a finite union of principal ideals, and (4) R is a finite direct product of finite local rings, special principal ideal rings (SPIRs), and (one-dimensional) Noetherian domains in which every maximal ideal is a finite union of principal ideals (or equivalently, weak CK domains). We show that a domain R is a weak CK domain if and only if R is a Noetherian domain with R M a CK domain for each maximal ideal M of R and Pic(R) = 0.

Specific N-linked glycosylation sites modulate synthesis and secretion of von Willebrand factor

AbstractWe examined the role that N-linked glycans play in the synthesis and expression of von Willebrand Factor (VWF). Blocking the addition of N-linked glycans (NLGs) or inhibiting initial glycan processing prevented secretion of VWF. To determine whether specific glycosylation sites were important, the 16 VWF N-linked glycosylation sites were mutated followed by expression in HEK293T cells. Four NLG mutants affected VWF expression: N99Q (D1 domain), N857Q (D' domain), N2400Q (B1 domain), and N2790Q (CK domain) either abolished or reduced secretion of VWF and this was confirmed by metabolic labeling. Multimer analysis of mutant N2790Q cell lysate revealed an increase in VWF monomers, which was also observed when the isolated CK domain was expressed with N2790 mutated. Immunofluorescence microscopy showed that mutants N99Q, N857Q, and N2790Q were primarily retained within the ER, producing only few pseudo Weibel-Palade bodies over longer time periods compared with wtVWF. All the variants also showed an increase in free thiol reactivity. This was greatest with N857Q and D4-C2 NLG mutants, which had approximately 6-fold and 3- to 4-fold more free thiol reactivity than wtVWF. These data provide further evidence of the critical role that individual N-linked glycans play in determining VWF synthesis and expression.

Assembly, Storage, and Secretion of Von Willebrand Factor.

Von Willebrand factor (VWF) plays a central role in hemostasis, and dysregulation of VWF can cause either bleeding or thrombosis. Endothelial cells assemble VWF multimers in two stages that occur in distinct intracellular compartments: proVWF subunits dimerize in the endoplasmic reticulum through “tail-to-tail” disulfide bonds between C-terminal CK domains, and the proVWF dimers form enormous multimers in the Golgi through “head-to-head” disulfide bonds between N-terminal D3 domains. The finished multimers are packaged into ordered tubules within storage organelles called Weibel-Palade bodies (WPB), and tubular packing is necessary for the secretion of VWF filaments that have normal hemostatic function. We have recreated VWF tubule assembly in vitro, starting with pure VWF propeptide (domains D1D2) and disulfide-linked dimers of adjacent N-terminal D’D3 domains. No other cellular proteins or components of WPB are needed. Assembly requires low pH and calcium ions, similar to conditions in the Golgi. Quick-freeze deep-etch electron microscopy and three-dimensional reconstruction of negatively stained images show that tubules contain a repeating unit of one D’D3 dimer and two propeptides arranged in a right-handed helix with 4.2 units per turn. The symmetry and location of interdomain contacts suggest that decreasing pH along the secretory pathway coordinates the disulfide-linked assembly of VWF multimers with their tubular packaging. Secretion into the blood exposes VWF tubules to neutral pH conditions, releasing the constraints that maintain tubular packing and permitting the orderly unfurling of large VWF multimers. Some VWF multimers bind platelets and initiate the growth of platelet-rich thrombi. Under normal circumstances, these thrombi are limited in size by ADAMTS13, a metalloprotease that cleaves VWF multimers and releases the platelets. The absence of large VWF multimers causes bleeding that is typical of von Willebrand disease. Conversely, congenital or acquired deficiency of ADAMTS13 prevents the dissolution of VWF-platelet aggregates, which can cause the widespread microvascular thrombosis that characterizes thrombotic thrombocytopenic purpura (TTP). Interestingly, ADAMTS13 deficiency alone need not trigger TTP. Some patients persist for months or years without active disease but become ill whenever they suffer additional inflammatory stress associated with infection, surgery, or pregnancy. Thus, interactions between inflammatory mediators and endothelial cells can determine the course of VWF-dependent thrombosis. The mechanisms responsible for these phenomena remain poorly understood.

Molecular Background of “Smeary” von Willebrand Factor Multimers.

Background: Multimer analysis of von Willebrand factor (VWF) is a most important technique to classify patients with von Willebrand disease (VWD) type 2. Besides “classical” multimer patterns a “smeary” appearance of individual VWF oligomers is increasingly observed and has previously been regarded as a pre-analytical artifact.Objective: To phenotypically and genotypically assess the molecular background of “smeary” VWF multimers.Patients and methods: Samples of 8 VWD patients were analyzed in our reference lab (UB) for further classification and molecular testing. Multimer profiles were assessed by intermediate resolution gels. VWF:CB and VWF:GpIb binding were used as functional assays. VWF gene mutation analysis was performed in all index cases (IC). The causal relationship between genotype and phenotype was studied by analyzing recombinant mutants in comparison to wildtype VWF.Results: In all IC the phenotype correlated with particular mutations in the VWF D3 domain (G1172D), the A1 domain (R1315C, R1374S, R1374C, R1399C), the D4 domain (C2257R), the C1 domain (R2464C) and in the region close to the CK domain (C2671Y), respectively. The multimer patterns of recombinant mutant VWF was of a “smeary” appearance and closely resembled those of mutant plasma VWF. Mutations in the A1 domain additionally correlated with severe GpIb binding deficiency.Conclusions: Our data suggest a molecular cause of the “smeary” multimer structure rather than pre-analytical artifacts. Most of the mutations identified involved cysteine residues suggesting an influence on the VWF secondary structure which is determined by intra- and intermolecular disulfide bonds. This could explain the peculiar multimer appearance. The functional deficits, however, seem to depend on the location of the mutations with a significant impact on GpIb binding of mutants in the A1 domain.

Two Cys residues essential for von Willebrand factor multimer assembly in the Golgi

Von Willebrand factor (VWF) dimerizes through C-terminal CK domains, and VWF dimers assemble into multimers in the Golgi by forming intersubunit disulfide bonds between D3 domains. This unusual oxidoreductase reaction requires the VWF propeptide (domains D1D2), which acts as an endogenous pH-dependent chaperone. The cysteines involved in multimer assembly were characterized by using a VWF construct that encodes the N-terminal D1D2D'D3 domains. Modification with thiol-specific reagents demonstrated that secreted D'D3 monomer contained reduced Cys, whereas D'D3 dimer and propeptide did not. Reduced Cys in the D'D3 monomer were alkylated with N-ethylmaleimide and analyzed by mass spectrometry. All 52 Cys within the D'D3 region were observed, and only Cys(1099) and Cys(1142) were modified by N-ethylmaleimide. When introduced into the D1D2D'D3 construct, the mutation C1099A or C1142A markedly impaired the formation of D'D3 dimers, and the double mutation prevented dimerization. In full-length VWF, the mutations C1099A and C1099A/C1142A prevented multimer assembly; the mutation C1142A allowed the formation of almost exclusively dimers, with few tetramers and no multimers larger than hexamers. Therefore, Cys(1099) and Cys(1142) are essential for the oxidoreductase mechanism of VWF multimerization. Cys(1142) is reported to form a Cys(1142)-Cys(1142) intersubunit bond, suggesting that Cys(1099) also participates in a Cys(1099)-Cys(1099) disulfide bond between D3 domains. This arrangement of intersubunit disulfide bonds implies that the dimeric N-terminal D'D3 domains of VWF subunits align in a parallel orientation within VWF multimers.

Clearance mechanisms of von Willebrand factor and factor VIII.

Clearance mechanisms of von Willebrand factor and factor VIII.

Impaired dimerization of von Willebrand factor subunit due to mutation A2801D in the CK domain results in a recessive type 2A subtype IID von Willebrand disease

The CK domain of von Willebrand factor (VWF) is involved in the dimerization of the protein. We identified the homozygous substitution A2801D of the CK domain in two siblings. Patients had low levels of VWF in plasma, abnormal ristocetin-induced binding to platelets and abnormal multimeric pattern with a lack of high molecular weight (HMW) forms and the presence of intervening bands between normal multimers. Accordingly, they were classified in type 2A, subtype IID, von Willebrand disease (VWD). Both asymptomatic parents carried the mutation at the heterozygous state. Their plasmaVWF exhibited the full range of multimers found in normal plasma. When analyzed by high resolution gel electrophoresis, very faint bands corresponding to the position of intervening bands of the propositus can be observed. The mutated recombinant (r)VWF-D2801, the hybrid rVWF-A/D2801 and the mutated C-terminal VWF fragment rSPII-D2801 were expressed in COS-7 cells. rVWF-D2801 showed an abnormal multimeric distribution similar to that of the propositus'VWF with intervening bands and a lack of HMW species. rVWF-A/D2801 exhibited the full range of multimers and the aberrant sized forms observed both in propositus'VWF and in rVWF-D2801. rSPII-WT assembled correctly into a dimer of 220 kDa. rSPII-D2801 appeared as a mixture of monomeric and dimeric forms which may be related to the abnormal multimeric pattern of the propositus and both mutated rVWF. We concluded that mutation A2801D disturbs the folding of the CK domain, which may result in a mixture of monomers and dimers of VWF. Multimers containing either an odd or even number of mature subunits are produced, and the presence of monomers appears to limit the degree of multimerization. In the heterozygousVWF, the presence of normal dimers improves the multimerization process. In conclusion, the mutation A2801D appears to be responsible for a recessive type 2A, subtype IID, VWD.

Type 3 Von Willebrand Disease Caused by Homozygous Splicing out of Exon 50.

Von Willebrand disease (VWD), a congenital bleeding disorder, results from quantitative or qualitative defects of von Willebrand factor (VWF). Overall, the estimated prevalence for VWD could be as high as 1.3%, although variable penetrance makes accurate estimation difficult.Quantitative defects of VWF result in disorders classified as Type 1 (Mild- moderate reductions in VW:Ag, VW:C and FVIII levels) and Type 3 (very low levels or absence of platelet and plasma VWF). Type 1 VWD typically has an autosomal dominant mode of inheritance, but its diagnosis is complicated by reduced penetrance and variable expression. Type 3 VWD is classified as having an autosomal recessive mode of inheritance, and may result from a homozygous expression of Type 1. The bleeding diathesis is characteristically severe in this group of patients.VWF levels are strongly associated with ABO group, and this can make diagnosis of type 1 VWD on the basis of phenotypic data difficult. This, and the variability in clinical symtoms should make molecular diagnosis of VWD a useful tool.The VWF gene is large, 52 exons over approximately 178kb, and codes for a protein comprised of 2813 amino acids. The gene is highly polymorphic, and there is a pseudo-gene with 97% homology to exons 23–34 of the functional gene. These factors combine to present difficulties in analysis of the VWF gene solely at the DNA level.We present a consanguinous family which exhibit type 1 and type 3 von Willebrand Disease (VWD). The parents, who are second cousins, both have type 1VWD. Of their four daughters, one has a normal VW phenotype, one has type 1 VWD, and two are phenotypically and clinically characteristic of Type 3 VWD. Sequencing all 52 exons of the VW gene identiifed a T→A substitution at position IVS 50+5. This was the only base change found that had not been not previously identified as a polymorphism. However, changes within the consensus splice sequence do not necessarily represent causative mutations. To support the fact that this change did represent the VWF defect in this family we demonstrated that it segregated with the disease, and was not found in any of 120 normal alleles tested, thus was not a common polymorphism. Comparison with the consensus donor splice sequence (ATGTGAGT) showed that the IVS 50 wild type sequence (AGGTAAGT) is a less than perfect fit. The introduction of a further alteration into this splice junction increases the likelihood of aberrant splicing. This potential was also highlighted by the use of splice site prediction software.The value of DNA analysis in predicting splice variants is limited, as apart from changes at the conserved bases there is naturally occuring variation. RNA analysis is necessary to demonstrate that splice junction variants are effective mutations. RNA analysis of the two Type 3 VWD sisters revealed that exon 50 is spliced out of the mRNA. This was surprising as abolition of a donor splice juction would be expected to cause either the inclusion of intronic sequence or the skipping of the next exon ( in this case exon 51).Exon 50 lies within the CK domain, which comprises the last 150 amino acids of the mature protein. These are essential for the dimerisation of the VWF subunits, which occurs within the endoplasmic reticulum (ER), and is a vital precursor to multimer formation in the Golgi apparatus.There have been several reports of possible splice defects in the VWF gene. However, few have been analysed at the RNA level to elucidate their effect. This family clearly demonstrates the benefits of utilising a RNA based strategy for the analysis of the VWF gene.

Amino Acid Sequence of a-Subunit in Hen Egg White Ovomucin Deduced from Cloned cDNA

The primary amino acid sequence of alpha-subunit in ovomucin (OVM) from hen thick egg white was determined. The 2087 amino acid residues with a relative molecular mass of 230.9 kDa along the full length of the alpha-subunit were represented. The alpha-subunit contains domains, arranged from the N- to C-terminals in the following order: D1-D2-D'-D3-R (central region)-D4-C1-CK (Cystine-knot), in a manner similar to the arrangement of D, C and CK domains in human pre-pro-von Willebrand factor (hpp-vWF) and hMUC2. The alpha-subunit showed identities on amino acid sequences with hpp-vWF and hMUC2 at 33 and 41% in the N-terminal region and 30 and 38% in the C-terminal region, respectively. The numbers and positions of cysteine residues were highly conserved among alpha-subunit, hpp-vWF and hMUC2. However, R showed no virtual sequence homology with the corresponding regions in two proteins. It was estimated that alpha-subunit was not part of a large peptide of OVM, but was independently synthesized from beta-subunit.

The complete genomic organization of the human MUC6 and MUC2 mucin genes

The complete genomic organization of the two mucin genes MUC2 and MUC6 was obtained by comparison of new and published mRNA sequences with newly available human genomic sequence. The two genes are located 38.5 kb apart in a head-to-head orientation within a gene complex on chromosome 11p15.5. The N-terminal organization of MUC6 is highly similar to that of MUC2, containing the D1, D2, D′, and D3 Von Willebrand factor domains followed by the large tandem repeat domains located in exons 31 and 30, respectively. MUC6 has a much smaller C-terminal domain (101 amino acids) encoded by 2 exons containing only the CK domain, compared with MUC2, which has a C-terminal domain of 859 amino acids containing the D4, C, D, and CK domains, encoded by 19 exons. The gene structures agreed partially but not completely with predictions from gene prediction programs.