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1
Richards, Allan J., e Martin P. Snead. "Molecular Basis of Pathogenic Variants in the Fibrillar Collagens". Genes 13, n. 7 (4 luglio 2022): 1199. http://dx.doi.org/10.3390/genes13071199.
Abstract (sommario):
The fibrillar collagen family is comprised of the quantitatively major types I, II and III collagens and the quantitatively minor types V and XI. These form heterotypic collagen fibrils (composed of more than a single collagen type) where the minor collagens have a regulatory role in controlling fibril formation and diameter. The structural pre-requisites for normal collagen biosynthesis and fibrillogenesis result in many places where this process can be disrupted, and consequently a wide variety of phenotypes result when pathogenic changes occur in these fibrillar collagen genes. Another contributing factor is alternative splicing, both naturally occurring and as the result of pathogenic DNA alterations. This article will discuss how these factors should be taken into account when assessing DNA sequencing results from a patient.
2
Koch, M., B. Bohrmann, M. Matthison, C. Hagios, B. Trueb e M. Chiquet. "Large and small splice variants of collagen XII: differential expression and ligand binding." Journal of Cell Biology 130, n. 4 (15 agosto 1995): 1005–14. http://dx.doi.org/10.1083/jcb.130.4.1005.
Abstract (sommario):
Collagen XII has a short collagenous tail and a very large, three-armed NC3 domains consisting primarily of fibronectin type III repeats. Differential splicing within this domain gives rise to a large (320 kD) and a small (220 kD) subunit; the large but not the small can carry glycosaminoglycan. To investigate whether collagen XII variants have distinct expression patterns and functions, we generated antibody and cDNA probes specific for the alternatively spliced domain. We report here that the large variant has a more restricted expression in embryonic tissue than the small. For example, whereas the small variant is widespread in the dermis, the large is limited to the base of feather buds. Distinct proportions of mRNA for the two variants were detected depending on the tissue. Monoclonal antibodies allowed us to separate collagen XII variants, and to show that homo- and heterotrimers exist. Collagen XII variants differ in ligand binding. Small subunits interact weakly with heparin via their COOH-terminal domain. Large subunits have additional, stronger heparin-binding site(s) in their NH2-terminal extra domain. In vivo, both large and small collagen XII are associated with interstitial collagen. Here we show biochemically and ultrastructurally that collagen XII can be incorporated into collagen I fibrils when it is present during, but not after, fibril formation. Removal of the collagenous domain of collagen XII reduces its coprecipitation with collagen I. Our results indicate that collagen XII is specifically associated with fibrillar collagen, and that the large variant has binding sites for extracellular ligands not present in the small variant.
3
Nishi, Akari, Hikaru Matsui, Azumi Hirata, Atsushi Mukaiyama, Shun-ichi Tanaka, Takuya Yoshizawa, Hiroyoshi Matsumura, Ryota Nomura, Kazuhiko Nakano e Kazufumi Takano. "Structure, Stability and Binding Properties of Collagen-Binding Domains from Streptococcus mutans". Chemistry 5, n. 3 (1 settembre 2023): 1911–20. http://dx.doi.org/10.3390/chemistry5030130.
Abstract (sommario):
Collagen-binding proteins (CBP), Cnm and Cbm, from Streptococcus mutans are involved in infective endocarditis caused by S. mutans because of their collagen-binding ability. In this study, we focused on the collagen-binding domain (CBD), which is responsible for the collagen-binding ability of CBP, and analyzed its structure, binding activity, and stability using CBD domain variants. The CBD consists of the N1 domain, linker, N2 domain, and latch (N1-N2~) as predicted from the amino acid sequences. The crystal structure of the Cnm/CBD was determined at a 1.81 Å resolution. N1_linker_N2 forms a ring structure that can enfold collagen molecules, and the latch interacts with N1 to form a ring clasp. N1 and N2 have similar immunoglobulin folds. The collagen-binding activities of Cbm/CBD and its domain variants were examined using ELISA. N1-N2~ bound to collagen with KD = 2.8 μM, and the latch-deleted variant (N1-N2) showed weaker binding (KD = 28 μM). The linker-deleted variant (N1N2~) and single-domain variants (N1 and N2) showed no binding activity, whereas the domain-swapped variant (N2-N1~) showed binding ability, indicating that the two N-domains and the linker are important for collagen binding. Thermal denaturation experiments showed that N1-N2 was slightly less stable than N1-N2~, and that N2 was more stable than N1. The results of this study provide a basis for the development of CBD inhibitors and applied research utilizing their collagen-binding ability.
4
Ritelli, Marco, Valeria Cinquina, Marina Venturini, Letizia Pezzaioli, Anna Formenti, Nicola Chiarelli e Marina Colombi. "Expanding the Clinical and Mutational Spectrum of Recessive AEBP1-Related Classical-Like Ehlers-Danlos Syndrome". Genes 10, n. 2 (12 febbraio 2019): 135. http://dx.doi.org/10.3390/genes10020135.
Abstract (sommario):
Ehlers-Danlos syndrome (EDS) comprises clinically heterogeneous connective tissue disorders with diverse molecular etiologies. The 2017 International Classification for EDS recognized 13 distinct subtypes caused by pathogenic variants in 19 genes mainly encoding fibrillar collagens and collagen-modifying or processing proteins. Recently, a new EDS subtype, i.e., classical-like EDS type 2, was defined after the identification, in six patients with clinical findings reminiscent of EDS, of recessive alterations in AEBP1, which encodes the aortic carboxypeptidase–like protein associating with collagens in the extracellular matrix. Herein, we report on a 53-year-old patient, born from healthy second-cousins, who fitted the diagnostic criteria for classical EDS (cEDS) for the presence of hyperextensible skin with multiple atrophic scars, generalized joint hypermobility, and other minor criteria. Molecular analyses of cEDS genes did not identify any causal variant. Therefore, AEBP1 sequencing was performed that revealed homozygosity for the rare c.1925T>C p.(Leu642Pro) variant classified as likely pathogenetic (class 4) according to the American College of Medical Genetics and Genomics (ACMG) guidelines. The comparison of the patient’s features with those of the other patients reported up to now and the identification of the first missense variant likely associated with the condition offer future perspectives for EDS nosology and research in this field.
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Flood, Veronica H., Abraham C. Schlauderaff, Paula M. Jacobi, Tricia L. Slobodianuk, Robert R. Montgomery, Sandra L. Haberichter e The Zimmerman Program Investigators. "VWF Interaction With Type IV Collagen Is Mediated Through Critical VWF A1 Domain Residues". Blood 122, n. 21 (15 novembre 2013): 29. http://dx.doi.org/10.1182/blood.v122.21.29.29.
Abstract (sommario):
Abstract Von Willebrand factor (VWF) plays a key role in coagulation by tethering platelets to injured subendothelium via binding sites for platelet glycoprotein Ib and collagen. The binding sites for types I and III collagen in the VWF A3 domain are well characterized, and defects in this region have been implicated in von Willebrand disease (VWD). Additional collagens present in the vasculature may also be involved in interactions with VWF. A VWF A1 sequence variation, p.R1399H, has been associated with decreased binding to type VI collagen, but the clinical significance of this observation remains unclear. Type IV collagen is a common component of the basement membrane and as such may be an important ligand for VWF. While some VWD testing utilizes types I or III collagen, current clinical testing does not include collagen IV or VI. To characterize the role of the VWF A1 domain in VWF-type IV collagen interactions, we generated several A1 domain variant human and/or murine recombinant VWF (rVWF) constructs including R1399H and several type 2M VWD variants localized to the same region (S1387I, Q1402P, and an 11 amino acid deletion mutant encompassing amino acids 1392-1402). These constructs were then expressed in HEK 293T cells. To further assess the role of the A1 domain, scanning alanine mutagenesis (SAM) of residues 1387 through 1412 was conducted. VWF antigen levels (VWF:Ag), collagen binding with type III (VWF:CB3), IV (VWF:CB4), or VI (VWF:CB6) collagen were determined, and multimer distribution was assessed for all recombinant VWF variants. The role of R1399H in the context of human rVWF was characterized initially. Although VWF:Ag, VWF:CB3, and multimer distribution were normal for R1399H compared to wild-type (WT VWF), VWF:CB4 was undetectable. To examine this effect in a mouse model, the R1399H variant was expressed in the context of murine rVWF and collagen binding was determined. Similar to the human variant, murine R1399H rVWF demonstrated significantly reduced binding to murine type IV collagen, at only 7% of the binding seen with WT murine rVWF. In order to examine the behavior of R1399H under shear conditions, either WT or R1399H murine rVWF DNA was hydrodynamically injected into the tail veins of VWF -/- mice to induce expression of the proteins; blood was drawn from the vena cava 24 hours later and then examined on the VenaFlux flow apparatus. VWF expression levels and multimer distribution were similar for the R1399H- and WT-injected mice. Under static conditions, the murine plasma-derived R1399H demonstrated decreased VWF:CB4, at only 16% of the levels seen with WT VWF. No defect was seen in VWF:CB3. Furthermore, when binding to type IV collagen was assessed under flow conditions by VenaFlux, platelet adhesion was significantly decreased in mice expressing R1399H VWF as compared to mice expressing WT VWF. When examining other A1 domain variants, Q1402P and del1392-1402 demonstrated absent VWF:CB4 while S1387I demonstrated a significant reduction in VWF:CB4 compared to WT VWF. All SAM VWF A1 domain variants demonstrated normal expression, multimerization, and VWF:CB3. However, type IV collagen binding was absent for R1392A, R1395A, R1399A, and K1406A and was reduced to less than 50% of WT VWF for Q1402A, K1405A, and K1407A. These residues map to an outside face of the VWF A1 domain crystal structure, and are likely the critical residues for VWF binding to type IV collagen. Taken together, these data demonstrate that the type IV collagen binding site localizes to a specific region of the VWF A1 domain. Mutations in this region of VWF may be clinically significant due to a defect in the ability of VWF to attract platelets to exposed type IV collagen which may contribute to bleeding symptoms seen in VWD. Disclosures: No relevant conflicts of interest to declare.
6
Shida, Yasuaki, Christine Brown, Jeff Mewburn, Kate Sponagle, Ozge Danisment, Barbara Vidal, Carol A. Heagadorn e David Lillicrap. "Comprehensive In Vitro and In Vivo Characterization of Loss and Gain-of-Function Von Willebrand Factor Collagen Binding Variants Using a Mouse Model System",. Blood 118, n. 21 (18 novembre 2011): 3266. http://dx.doi.org/10.1182/blood.v118.21.3266.3266.
Abstract (sommario):
Abstract Abstract 3266 Von Willebrand Factor (VWF) is a large multimeric glycoprotein that mediates platelet adhesion to the damaged blood vessel wall and subsequent platelet aggregation at the site of injury. Rare mutations in the VWF A3 domain, that disrupt collagen binding, have been found in patients with a mild bleeding phenotype. However, the analysis of these aberrant VWF-collagen interactions has been relatively limited. Thus, in this study, we have developed mouse models of collagen binding mutants and analyzed the function of the A3 and A1 domains using comprehensive in vitro and in vivo approaches. All of the collagen binding variant AAs are conserved in mice. 6 loss-of-function (S1731T, W1745C, S1783A, H1786D, A1 deletion, A3 deletion) and 1 gain-of-function (L1757A) variant was generated in the context of the mouse VWF cDNA. The 4 loss-of-function missense mutants have all been described in patients with mild bleeding phenotypes. The recombinant mouse VWFs (rmVWF) were synthesized in HEK293T cells and analyzed for type I and III collagen binding in both a static assay (CBA) and a flow-based assay at 2,500s−1 in which VWF is bound to collagen on a surface, and labeled platelet adhesion is quantified. The multimer profile of all the rmVWFs was normal. The expression level of the rmVWF derived from HEK293T cells was quantified. W1745C and the A3 deletion showed significantly lower levels of expression and the A1 deletion mutant showed strong intracellular retention. In the static collagen binding assay, S1731T showed almost normal binding to collagen type I and a 50% reduction in binding to collagen type III. The other 3 missense variants, W1745C, S1783A and H1786D, showed reduced binding to both collagens I and III, and the A3 deletion mutant showed absent binding. In the in vitro flow assay, the sensitivity to detect defects in collagen binding was superior to the static assay, although the patterns of binding defects were similar. W1745C showed similar low levels of platelet adhesion to both types of collagen, while S1783A and H1786D showed a lack of platelet binding on the collagen III surface similar to the A3 deletion mutant, and a reduced binding to collagen type I similar to W1745C. The gain-of-function mutant showed consistent enhanced collagen binding and platelet adhesion in the static and flow assays, respectively. In vivo studies delivered the mVWF cDNAs with a strong liver specific promoter by hydrodynamic injection. At 7 days post-delivery, the VWF:Ag levels in the WT and collagen binding variant mice were similar, apart from the W1745C mutant, that showed 14.6% levels compared to WT. Platelet counts and multimer patterns were normal with the collagen binding variants. In vivo intravital microscopy studies were performed using the cremaster arteriolar model when VWF levels were in a physiological range. Thrombosis was induced by 10%FeCl3 applied for 3 mins. Platelets were labeled in vivo by Rhodamine 6G and the thrombus development was analyzed by spinning disc confocal microscopy. Loss-of-function mutants showed transient platelet adhesion at the site of injury, however the adhesion was unstable and vessel occlusion was not observed. Using three complementary experimental systems we have been able to confirm the collagen binding defects in this group of variant VWFs. There is a differential sensitivity to the two forms of collagen and of the three experimental systems. The A3 deletion mutant consistently resulted in the most severe phenotype while the missense mutants showed variable degrees of functional deficit. Disclosures: No relevant conflicts of interest to declare.
7
Mikhail, Kristen A., Elizabeth VanSickle e Linda Z. Rossetti. "Milder presentation of osteogenesis imperfecta type VIII due to compound heterozygosity for a predicted loss-of-function variant and novel missense variant inP3H1—further expansion of the phenotypic spectrum". Molecular Case Studies 9, n. 1 (febbraio 2023): a006260. http://dx.doi.org/10.1101/mcs.a006260.
Abstract (sommario):
Osteogenesis imperfecta (OI) is a heritable disorder of bone metabolism characterized by multiple fractures with minimal trauma. Autosomal recessive OI type VIII is associated with biallelic pathogenic variants inP3H1and classically characterized by skeletal anomalies in addition to significant bone fragility, sometimes presenting with in utero fractures and/or neonatal lethality.P3H1encodes a collagen prolyl hydroxylase that critically 3-hydroxylates proline residue 986 on the α chain of collagen types I and II to achieve proper folding and assembly of mature collagen and is present in a complex with CRTAP and CypB. Most individuals with OI type VIII have had biallelic predicted loss-of-function variants leading to reduced or absent levels ofP3H1mRNA. The reported missense variants have all fallen in the catalytic domain of the protein and are thought to be associated with a milder phenotype. Here, we describe an infant presenting with five long bone fractures in the first year of life found to have a novel missense variant intranswith a nonsense variant inP3H1without any other bony anomalies on imaging. We hypothesize that missense variants in the catalytic domain of P3H1 lead to decreased but not absent hydroxylation of Pro986, with preserved KDEL retention signal and complex stability, causing an attenuated phenotype.
8
López-Márquez, Arístides, Matías Morín, Sergio Fernández-Peñalver, Carmen Badosa, Alejandro Hernández-Delgado, Daniel Natera-de Benito, Carlos Ortez et al. "CRISPR/Cas9-Mediated Allele-Specific Disruption of a Dominant COL6A1 Pathogenic Variant Improves Collagen VI Network in Patient Fibroblasts". International Journal of Molecular Sciences 23, n. 8 (16 aprile 2022): 4410. http://dx.doi.org/10.3390/ijms23084410.
Abstract (sommario):
Collagen VI-related disorders are the second most common congenital muscular dystrophies for which no treatments are presently available. They are mostly caused by dominant-negative pathogenic variants in the genes encoding α chains of collagen VI, a heteromeric network forming collagen; for example, the c.877G>A; p.Gly293Arg COL6A1 variant, which alters the proper association of the tetramers to form microfibrils. We tested the potential of CRISPR/Cas9-based genome editing to silence or correct (using a donor template) a mutant allele in the dermal fibroblasts of four individuals bearing the c.877G>A pathogenic variant. Evaluation of gene-edited cells by next-generation sequencing revealed that correction of the mutant allele by homologous-directed repair occurred at a frequency lower than 1%. However, the presence of frameshift variants and others that provoked the silencing of the mutant allele were found in >40% of reads, with no effects on the wild-type allele. This was confirmed by droplet digital PCR with allele-specific probes, which revealed a reduction in the expression of the mutant allele. Finally, immunofluorescence analyses revealed a recovery in the collagen VI extracellular matrix. In summary, we demonstrate that CRISPR/Cas9 gene-edition can specifically reverse the pathogenic effects of a dominant negative variant in COL6A1.
9
Zhytnik, Lidiia, Binh Ho Duy, Marelise Eekhoff, Lisanne Wisse, Gerard Pals, Ene Reimann, Sulev Kõks et al. "Phenotypic Variation in Vietnamese Osteogenesis Imperfecta Patients Sharing a Recessive P3H1 Pathogenic Variant". Genes 13, n. 3 (24 febbraio 2022): 407. http://dx.doi.org/10.3390/genes13030407.
Abstract (sommario):
Osteogenesis imperfecta (OI) is a syndromic disorder of bone fragility with high variation in its clinical presentation. Equally variable is molecular aetiology; recessive forms are caused by approximately 20 different genes, many of which are directly implicated in collagen type I biosynthesis. Biallelic variants in prolyl 3-hydroxylase 1 (P3H1) are known to cause severe OI by affecting the competence of the prolyl 3-hydroxylation—cartilage associated protein—peptidyl-prolyl cis-trans isomerase B (P3H1-CRTAP-CyPB) complex, which acts on the Pro986 residue of collagen type I α 1 (COL1A1) and Pro707 collagen type I α 2 (COL1A2) chains. The investigation of an OI cohort of 146 patients in Vietnam identified 14 families with P3H1 variants. The c.1170+5G>C variant was found to be very prevalent (12/14) and accounted for 10.3% of the Vietnamese OI cohort. New P3H1 variants were also identified in this population. Interestingly, the c.1170+5G>C variants were found in families with the severe clinical Sillence types 2 and 3 but also the milder types 1 and 4. This is the first time that OI type 1 is reported in patients with P3H1 variants expanding the clinical spectrum. Patients with a homozygous c.1170+5G>C variant shared severe progressively deforming OI type 3: bowed long bones, deformities of ribcage, long phalanges and hands, bluish sclera, brachycephaly, and early intrauterine fractures. Although it remains unclear if the c.1170+5G>C variant constitutes a founder mutation in the Vietnamese population, its prevalence makes it valuable for the molecular diagnosis of OI in patients of the Kinh ethnicity. Our study provides insight into the clinical and genetic variation of P3H1-related OI in the Vietnamese population.
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Micale, Lucia, Silvia Morlino, Annalisa Schirizzi, Emanuele Agolini, Grazia Nardella, Carmela Fusco, Stefano Castellana et al. "Exon-Trapping Assay Improves Clinical Interpretation of COL11A1 and COL11A2 Intronic Variants in Stickler Syndrome Type 2 and Otospondylomegaepiphyseal Dysplasia". Genes 11, n. 12 (17 dicembre 2020): 1513. http://dx.doi.org/10.3390/genes11121513.
Abstract (sommario):
Stickler syndrome (SS) is a hereditary connective tissue disorder affecting bones, eyes, and hearing. Type 2 SS and the SS variant otospondylomegaepiphyseal dysplasia (OSMED) are caused by deleterious variants in COL11A1 and COL11A2, respectively. In both genes, available database information indicates a high rate of potentially deleterious intronic variants, but published evidence of their biological effect is usually insufficient for a definite clinical interpretation. We report four previously unpublished intronic variants in COL11A1 (c.2241 + 5G>T, c.2809 − 2A>G, c.3168 + 5G>C) and COL11A2 (c.4392 + 1G>A) identified in type 2 SS/OSMED individuals. The pathogenic effect of these variants was first predicted in silico and then investigated by an exon-trapping assay. We demonstrated that all variants can induce exon in-frame deletions, which lead to the synthesis of shorter collagen XI α1 or 2 chains. Lacking residues are located in the α-triple helical region, which has a crucial role in regulating collagen fibrillogenesis. In conclusion, this study suggests that these alternative COL11A1 and COL11A2 transcripts might result in aberrant triple helix collagen. Our approach may help to improve the diagnostic molecular pathway of COL11-related disorders.
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Bruni, Valentina, Cristina Barbara Spoleti, Andrea La Barbera, Vincenzo Dattilo, Emma Colao, Carmela Votino, Emanuele Bellacchio, Nicola Perrotti, Sabrina Giglio e Rodolfo Iuliano. "A Novel Splicing Variant of COL2A1 in a Fetus with Achondrogenesis Type II: Interpretation of Pathogenicity of In-Frame Deletions". Genes 12, n. 9 (10 settembre 2021): 1395. http://dx.doi.org/10.3390/genes12091395.
Abstract (sommario):
Achondrogenesis type II (ACG2) is a lethal skeletal dysplasia caused by dominant pathogenic variants in COL2A1. Most of the variants found in patients with ACG2 affect the glycine residue included in the Gly-X-Y tripeptide repeat that characterizes the type II collagen helix. In this study, we reported a case of a novel splicing variant of COL2A1 in a fetus with ACG2. An NGS analysis of fetal DNA revealed a heterozygous variant c.1267-2_1269del located in intron 20/exon 21. The variant occurred de novo since it was not detected in DNA from the blood samples of parents. We generated an appropriate minigene construct to study the effect of the variant detected. The minigene expression resulted in the synthesis of a COL2A1 messenger RNA lacking exon 21, which generated a predicted in-frame deleted protein. Usually, in-frame deletion variants of COL2A1 cause a phenotype such as Kniest dysplasia, which is milder than ACG2. Therefore, we propose that the size and position of an in-frame deletion in COL2A1 may be relevant in determining the phenotype of skeletal dysplasia.
12
Haberichter, Sandra L., David A. Jakab e Paula M. Jacobi. "Upstream Mechanisms Causing Type 1C Von Willebrand Disease (VWD): Contribution Of Defective Von Willebrand Factor (VWF) Multimerization, Regulated Storage, and Secretion". Blood 122, n. 21 (15 novembre 2013): 3571. http://dx.doi.org/10.1182/blood.v122.21.3571.3571.
Abstract (sommario):
Abstract One mechanism causing type 1 VWD is the reduced survival of VWF in plasma (type 1C VWD), characterized by markedly decreased VWF:Ag and VWF half-life, essentially normal multimers, increased ratio of VWF propeptide (VWFpp) to VWF:Ag, robust response to DDAVP, and normal ratios of VWF:CB, FVIII, or VWF:RCo to VWF:Ag. We enrolled 502 index cases with a pre-existing diagnosis of type 1 VWD through the Zimmerman Program for the Molecular and Clinical Biology of VWD. We confirmed 262 of the index cases as type 1 VWD (VWF:Ag or VWF:RCo ≤ 40 IU/dL). Of these, 58 met the criteria for type 1C VWD with VWFpp/VWF:Ag ≥ 3 and VWF:Ag ≤ 30 IU/dL. Sequence variations were identified in the VWF D3, A1, A2, and D4 domains. Little is known regarding the mechanisms causing type 1C VWD, but it has been assumed that VWF undergoes normal intracellular processing and secretion with rapid clearance upon release into plasma. We hypothesized that defective intracellular processing may contribute to the type 1C phenotype. We studied 10 type 1C variants including C1130Y, W1144G, R1205H, N1231S, R1315C, V1411E, R1527W, N2041S, Y2160C, and S2179F. Variants were expressed alone (homozygously) or with wild-type (WT) VWF (heterozygously) in HEK293T cells and VWF secretion, multimer structure, and binding to collagen (types III and VI), GPIb-alpha, and FVIII was analyzed. To assess regulated storage, variants were expressed homozygously in HEK293 cells where WT VWF forms elongated pseudo-Weibel-Palade bodies (pWPB). Five variants (C1130Y, R1315C, V1411E, N2041S, Y2160C) had severely decreased secretion and defective multimerization when homozygously expressed. These variants did not form pWPB, but appeared to co-localize with the endoplasmic reticulum, consistent with the severely impaired secretion. One variant, W1144G, had mildly reduced secretion, formed only dimeric VWF, and unexpectedly did not form pWPB. These multimer defective variants demonstrated decreased collagen binding and GPIb-alpha binding as would be predicted. The remaining variants (R1205H, N1231S, R1527W, S2179F) were normally secreted, multimerized, stored in pWPB, and had normal binding to FVIII, collagen, and GPIb-alpha. Interestingly, FVIII binding to homozygous VWF D3 variants C1130Y and W1144G was substantially reduced. This result is not entirely unexpected as the FVIII binding region in VWF has been mapped to the D’-D3 region. Co-expression with WT VWF essentially corrected defective secretion, although some variants still had moderately reduced secretion. Multimer structure appeared normal for all heterozygous variants, although staining which discriminates between variant and WT alleles revealed that for some variants, little variant VWF was actually expressed when transfected at a 1:1 ratio with WT. In sum, when variants were homozygously expressed, we observed a constellation of processing and functional defects. Only R1205H, N1231S, R1527W, and S2179F variants demonstrated normal processing and function. Heterozygous expression (consistent with patients) corrected most of the observed defects, although reduced secretion persisted for a subset of variants. We can conclude that while reduced plasma survival of VWF is a major determinant of the type 1C phenotype, additional upstream processing defects may contribute to the severity of the overall VWD phenotype. Disclosures: No relevant conflicts of interest to declare.
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Sivapalaratnam, Suthesh, Hayman Melissa, Claire Lentaigne, Melissa Chan, Marilena Crescente, Harriet Allan, Katherine Wedderburn et al. "Congenital Aspirin-like Defect As a Result of Autosomal Recessive Variants in PTGS1". Blood 132, Supplement 1 (29 novembre 2018): 1156. http://dx.doi.org/10.1182/blood-2018-99-118958.
Abstract (sommario):
Abstract Inherited defects of platelet function disorders are rare and difficult to diagnose due to lack of standardized platelet tests. An aspirin-like platelet defect is characterised by reduced thromboxane A2 (TXA2) signalling due to a defect in the arachidonic acid (AA) pathway in platelets. Patients with aspirin-like defect present with mild to moderate bleeding symptoms and impaired platelet aggregation responses to AA and ADP. This is similar to the irreversible effect of aspirin on platelets, which is mediated through inhibition of prostaglandin H synthase-1 also known as cyclooxygenase-1 (PTGS1/COX1). We for the first time report platelet function disorders due to autosomal recessive inheritance of variants in PTGS1. In a total of 3563 cases with bleeding disorders, comprising 1169 whole genome sequenced probands of the BRIDGE-BPD study and 2394 panel sequenced index cases of the ThromboGenomics cohort, we identified 15 unrelated cases of each cohort with an aspirin-like platelet function defect. Two of these cases had rare a variants in PTGS1, the gene encoding COX-1, which catalyses the conversion of arachidonic acid to prostaglandin H2. The first case presented with epistaxis and peri-operative bleeding. She had reduced platelet aggregation responses to arachidonic acid, ADP, collagen and epinephrine. Incubation of control blood with collagen resulted in enhanced levels of thromboxane B2, PGD2, PGE2, 11-HETE and 15-HETE which was absent in the index case.We identified a homozygous missense variant in PTGS1, p.Trp322Ser with a Combined Annotation Dependant Depletion Score (CADD) of 31.0. This variant was absent from GnomAD. The variant co-segregated in an autosomal recessive inheritance mode, with aspirin-like defect phenotype in the seven family members who were investigated. PTGS1 was not expressed on the platelets by western blot. Immunophenotyping demonstrated absence on the platelet surface but presence on neutrophils. The second case of the presented with menorrhagia, nosebleeds, easy bleeding and bruising. She had reduced aggregation responses to arachidonic acid, ADP, collagen and epinephrine. We identified two variants in cis: a splice-donor variant (g. 125133553 T>A), CADD 24.3; and an upstream non-coding variant (g. 125132069 C>G), CADD 16.63. The frequency of these variants were respectively; 1.7 x 10-5 and 8 x 10-3 in GnomAD. Platelet RNA and protein expression studies in the propositus revealed alternative splicing with the generation of a smaller protein due the splice variant. In contrast, the non coding variant had no effect on promoter or enhancer activity and therefore, is likely benign. In this case, the mode of inheritance is autosomal dominant with a dominant negative effect, which has been reported previously. For the other 13 cases of the Bridge-BPD study we also interrogated the non-coding space and interactors in the arachidonic acid pathway, none of which had genetic variants explaining the phenotype. For the 15 ThromboGenomics cohort cases because they were sequenced on targeted platform similar investigations could not take place. These cases could have a non inherited cause for the platelet defect or it is also permissible that variation in a hitherto undefined pathways unique to individual cases might be causal. In conclusion, we for the first time report autosomal recessive inheritance of variants in PTGS1 as cause for a rare inherited bleeding disorder. The effect of the mutation are selective loss of expression of PTGS1 within platelets and decreased enzyme function. Two previous reports demonstrated autosomal dominant inheritance. The first demonstrated autosomal dominant inheritance of variants in PTGS1 as modifier in a well characterized family with haemophilia A and platelet function disorder (Nance et al JTH 2016). The second reported rare heterozygous variants in PTGS1 in two cases with a bleeding tendency which was not further specified in the report (Bastida et al Haematologica 2018). Disclosures Laffan: Pfizer: Honoraria; Roche: Consultancy, Speakers Bureau.
14
Jäälinoja, Juha, Joni Ylöstalo, William Beckett, David J. S. Hulmes e Leena Ala-Kokko. "Trimerization of collagen IX α-chains does not require the presence of the COL1 and NC1 domains". Biochemical Journal 409, n. 2 (21 dicembre 2007): 545–54. http://dx.doi.org/10.1042/bj20070984.
Abstract (sommario):
Collagen IX is a heterotrimer of three α-chains, which consists of three COL domains (collagenous domains) (COL1–COL3) and four NC domains (non-collagenous domains) (NC1–NC4), numbered from the C-terminus. Although collagen IX chains have been shown to associate via their C-terminal NC1 domains and form a triple helix starting from the COL1 domain, it is not known whether chain association can occur at other sites and whether other collagenous and non-collagenous regions are involved. To address this question, we prepared five constructs, two long variants (beginning at the NC4 domain) and three short variants (beginning at the COL2 domain), all ending at the NC2 domain (or NC2 replaced by NC1), to study association and selection of collagen IX α-chains. Both long variants were able to associate with NC1 or NC2 at the C-terminus and form various disulfide-bonded trimers, but the specificity of chain selection was diminished compared with full-length chains. Trimers of the long variant ending at NC2 were shown to be triple helical by CD. Short variants were not able to assemble into disulfide-bonded trimers even in the presence of both conserved cysteine residues from the COL1–NC1 junction. Our results demonstrate that collagen IX α-chains can associate in the absence of COL1 and NC1 domains to form a triple helix, but the COL2–NC2 region alone is not sufficient for trimerization. The results suggest that folding of collagen IX is a co-operative process involving multiple COL and NC domains and that the COL1–NC1 region is important for chain specificity.
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Hayashi, Kaichi, Takeaki Ikeuchi, Ryo Morishita, Jun Qian, Kenji Kojima, Teisuke Takita, Keisuke Tanaka, Shunji Hattori e Kiyoshi Yasukawa. "The roles of histidine and tyrosine residues in the active site of collagenase in Grimontia hollisae". Journal of Biochemistry 168, n. 4 (9 maggio 2020): 385–92. http://dx.doi.org/10.1093/jb/mvaa055.
Abstract (sommario):
Abstract Collagenase from the Grimontia hollisae strain 1706B (Ghcol) is a zinc metalloproteinase with the zinc-binding motif H492EXXH496. It exhibits higher collagen-degrading activity than the collagenase from Clostridium histolyticum, which is widely used in industry. We previously examined the pH and temperature dependencies of Ghcol activity; Glu493 was thought to contribute acidic pKa (pKe1), while no residue was assigned to contribute alkaline pKa (pKe2). In this study, we introduced nine single mutations at the His or Tyr residues in and near the active site. Our results showed that H412A, H485A, Y497A, H578A and H737A retained the activities to hydrolyze collagen and gelatin, while H426A, H492A, H496A and Y568A lacked them. Purification of active variants H412A, H485A, H578A and H737A, along with inactive variants H492A and H496A, were successful. H412A preferred (7-methoxycoumarin-4-yl)acetyl-L-Lys-L-Pro-L-Leu-Gly-L-Leu-[N3-(2,4-dinitrophenyl)-L-2,3-diaminopropionyl]-L-Ala-L-Arg-NH2 to collagen, while H485A preferred collagen to the peptide, suggesting that His412 and His485 are important for substrate specificity. Purification of the active variant Y497A and inactive variants H426A and Y568A were unsuccessful, suggesting that these three residues were important for stability. Based on the reported crystal structure of clostridial collagenase, Tyr568 of Ghcol is suggested to be involved in catalysis and may be the ionizable residue for pKe2.
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Savige, Judy, Helen Storey, Elizabeth Watson, Jens Michael Hertz, Constantinos Deltas, Alessandra Renieri, Francesca Mari et al. "Consensus statement on standards and guidelines for the molecular diagnostics of Alport syndrome: refining the ACMG criteria". European Journal of Human Genetics 29, n. 8 (15 aprile 2021): 1186–97. http://dx.doi.org/10.1038/s41431-021-00858-1.
Abstract (sommario):
AbstractThe recent Chandos House meeting of the Alport Variant Collaborative extended the indications for screening for pathogenic variants in the COL4A5, COL4A3 and COL4A4 genes beyond the classical Alport phenotype (haematuria, renal failure; family history of haematuria or renal failure) to include persistent proteinuria, steroid-resistant nephrotic syndrome, focal and segmental glomerulosclerosis (FSGS), familial IgA glomerulonephritis and end-stage kidney failure without an obvious cause. The meeting refined the ACMG criteria for variant assessment for the Alport genes (COL4A3–5). It identified ‘mutational hotspots’ (PM1) in the collagen IV α5, α3 and α4 chains including position 1 Glycine residues in the Gly-X-Y repeats in the intermediate collagenous domains; and Cysteine residues in the carboxy non-collagenous domain (PP3). It considered that ‘well-established’ functional assays (PS3, BS3) were still mainly research tools but sequencing and minigene assays were commonly used to confirm splicing variants. It was not possible to define the Minor Allele Frequency (MAF) threshold above which variants were considered Benign (BA1, BS1), because of the different modes of inheritances of Alport syndrome, and the occurrence of hypomorphic variants (often Glycine adjacent to a non-collagenous interruption) and local founder effects. Heterozygous COL4A3 and COL4A4 variants were common ‘incidental’ findings also present in normal reference databases. The recognition and interpretation of hypomorphic variants in the COL4A3–COL4A5 genes remains a challenge.
17
Castroflorio, Enrico, Ana Joaquina Pérez Berná, Arístides López-Márquez, Carmen Badosa, Pablo Loza-Alvarez, Mónica Roldán e Cecilia Jiménez-Mallebrera. "The Capillary Morphogenesis Gene 2 Triggers the Intracellular Hallmarks of Collagen VI-Related Muscular Dystrophy". International Journal of Molecular Sciences 23, n. 14 (11 luglio 2022): 7651. http://dx.doi.org/10.3390/ijms23147651.
Abstract (sommario):
Collagen VI-related disorders (COL6-RD) represent a severe form of congenital disease for which there is no treatment. Dominant-negative pathogenic variants in the genes encoding α chains of collagen VI are the main cause of COL6-RD. Here we report that patient-derived fibroblasts carrying a common single nucleotide variant mutation are unable to build the extracellular collagen VI network. This correlates with the intracellular accumulation of endosomes and lysosomes triggered by the increased phosphorylation of the collagen VI receptor CMG2. Notably, using a CRISPR-Cas9 gene-editing tool to silence the dominant-negative mutation in patients’ cells, we rescued the normal extracellular collagen VI network, CMG2 phosphorylation levels, and the accumulation of endosomes and lysosomes. Our findings reveal an unanticipated role of CMG2 in regulating endosomal and lysosomal homeostasis and suggest that mutated collagen VI dysregulates the intracellular environment in fibroblasts in collagen VI-related muscular dystrophy.
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Villar-Quiles, Rocío N., Sandra Donkervoort, Alix de Becdelièvre, Corine Gartioux, Valérie Jobic, A. Reghan Foley, Riley M. McCarty et al. "Clinical and Molecular Spectrum Associated with COL6A3 c.7447A>G p.(Lys2483Glu) Variant: Elucidating its Role in Collagen VI-related Myopathies". Journal of Neuromuscular Diseases 8, n. 4 (30 luglio 2021): 633–45. http://dx.doi.org/10.3233/jnd-200577.
Abstract (sommario):
Background: Dominant and recessive autosomal pathogenic variants in the three major genes (COL6A1-A2-A3) encoding the extracellular matrix protein collagen VI underlie a group of myopathies ranging from early-onset severe conditions (Ullrich congenital muscular dystrophy) to milder forms maintaining independent ambulation (Bethlem myopathy). Diagnosis is based on the combination of clinical presentation, muscle MRI, muscle biopsy, analysis of collagen VI secretion, and COL6A1-A2-A3 genetic analysis, the interpretation of which can be challenging. Objective: To refine the phenotypical spectrum associated with the frequent COL6A3 missense variant c.7447A>G (p.Lys2483Glu). Methods: We report the clinical and molecular findings in 16 patients: 12 patients carrying this variant in compound heterozygosity with another COL6A3 variant, and four homozygous patients. Results: Patients carrying this variant in compound heterozygosity with a truncating COL6A3 variant exhibit a phenotype consistent with COL6-related myopathies (COL6-RM), with joint contractures, proximal weakness and skin abnormalities. All remain ambulant in adulthood and only three have mild respiratory involvement. Most show typical muscle MRI findings. In five patients, reduced collagen VI secretion was observed in skin fibroblasts cultures. All tested parents were unaffected heterozygous carriers. Conversely, two out of four homozygous patients did not present with the classical COL6-RM clinical and imaging findings. Collagen VI immunolabelling on cultured fibroblasts revealed rather normal secretion in one and reduced secretion in another. Muscle biopsy from one homozygous patient showed myofibrillar disorganization and rimmed vacuoles. Conclusions: In light of our results, we postulate that the COL6A3 variant c.7447A>G may act as a modulator of the clinical phenotype. Thus, in patients with a typical COL6-RM phenotype, a second variant must be thoroughly searched for, while for patients with atypical phenotypes further investigations should be conducted to exclude alternative causes. This works expands the clinical and molecular spectrum of COLVI-related myopathies.
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Kantaputra, Piranit Nik, Salita Angkurawaranon, Worrachet Intachai, Chumpol Ngamphiw, Bjorn Olsen, Sissades Tongsima, Timothy C. Cox e James R. Ketudat Cairns. "A Founder Intronic Variant in P3H1 Likely Results in Aberrant Splicing and Protein Truncation in Patients of Karen Descent with Osteogenesis Imperfecta Type VIII". Genes 14, n. 2 (26 gennaio 2023): 322. http://dx.doi.org/10.3390/genes14020322.
Abstract (sommario):
One of the most important steps in post-translational modifications of collagen type I chains is the hydroxylation of carbon-3 of proline residues by prolyl-3-hydroxylase-1 (P3H1). Genetic variants in P3H1 have been reported to cause autosomal recessive osteogenesis imperfecta (OI) type VIII. Clinical and radiographic examinations, whole-exome sequencing (WES), and bioinformatic analysis were performed in 11 Thai children of Karen descent affected by multiple bone fractures. Clinical and radiographic findings in these patients fit OI type VIII. Phenotypic variability is evident. WES identified an intronic homozygous variant (chr1:43212857A > G; NM_022356.4:c.2055 + 86A > G) in P3H1 in all patients, with parents in each patient being heterozygous for the variant. This variant is predicted to generate a new “CAG” splice acceptor sequence, resulting in the incorporation of an extra exon that leads to a frameshift in the final exon and subsequent non-functional P3H1 isoform a. Alternative splicing of P3H1 resulting in the absence of functional P3H1 caused OI type VIII in 11 Thai children of Karen descent. This variant appears to be specific to the Karen population. Our study emphasizes the significance of considering intronic variants.
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Eicher, John D., Ming-Huei Chen, Achilleas N. Pitsillides, Honghuang Lin, Narayanan Veeraraghavan, Jennifer A. Brody, Ginger A. Metcalf et al. "Whole exome sequencing in the Framingham Heart Study identifies rare variation in HYAL2 that influences platelet aggregation". Thrombosis and Haemostasis 117, n. 06 (2017): 1083–92. http://dx.doi.org/10.1160/th16-09-0677.
Abstract (sommario):
SummaryInhibition of platelet reactivity is a common therapeutic strategy in secondary prevention of cardiovascular disease. Genetic and environmental factors influence inter-individual variation in platelet reactivity. Identifying genes that contribute to platelet reactivity can reveal new biological mechanisms and possible therapeutic targets. Here, we examined rare coding variation to identify genes associated with platelet reactivity in a population-based cohort. To do so, we performed whole exome sequencing in the Framingham Heart Study and conducted single variant and gene-based association tests against platelet reactivity to collagen, adenosine diphosphate (ADP), and epinephrine agonists in up to 1,211 individuals. Single variant tests revealed no significant associations (p<1.44×10–7), though we observed a suggestive association with previously implicated MRVI1 (rs11042902, p = 1.95×10–7). Using gene-based association tests of rare and low-frequency variants, we found significant associations of HYAL2 with increased ADP-induced aggregation (p = 1.07×10–7) and GSTZ1 with increased epinephrine-induced aggregation (p = 1.62×10–6). HYAL2 also showed suggestive associations with epinephrine-induced aggregation (p = 2.64×10–5). The rare variants in the HYAL2 gene-based association included a missense variant (N357S) at a known N-glycosylation site and a nonsense variant (Q406*) that removes a glycophosphatidylinositol (GPI) anchor from the resulting protein. These variants suggest that improper membrane trafficking of HYAL2 influences platelet reactivity. We also observed suggestive associations of AR (p = 7.39×10–6) and MAPRE1 (p = 7.26×10–6) with ADP-induced reactivity. Our study demonstrates that gene-based tests and other grouping strategies of rare variants are powerful approaches to detect associations in population-based analyses of complex traits not detected by single variant tests and possible new genetic influences on platelet reactivity.Supplementary Material to this article is available online at www.thrombosis-online.com.
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Kiener, Sarah, Heather Troyer, Daniel Ruvolo, Paula Grest, Sara Soto, Anna Letko, Vidhya Jagannathan et al. "Independent COL17A1 Variants in Cats with Junctional Epidermolysis Bullosa". Genes 14, n. 10 (22 settembre 2023): 1835. http://dx.doi.org/10.3390/genes14101835.
Abstract (sommario):
Epidermolysis bullosa (EB), characterized by defective adhesion of the epidermis to the dermis, is a heterogeneous disease with many subtypes in human patients and domestic animals. We investigated two unrelated cats with recurring erosions and ulcers on ear pinnae, oral mucosa, and paw pads that were suggestive of EB. Histopathology confirmed the diagnosis of EB in both cats. Case 1 was severe and had to be euthanized at 5 months of age. Case 2 had a milder course and was alive at 11 years of age at the time of writing. Whole genome sequencing of both affected cats revealed independent homozygous variants in COL17A1 encoding the collagen type XVII alpha 1 chain. Loss of function variants in COL17A1 lead to junctional epidermolysis bullosa (JEB) in human patients. The identified splice site variant in case 1, c.3019+1del, was predicted to lead to a complete deficiency in collagen type XVII. Case 2 had a splice region variant, c.769+5G>A. Assessment of the functional impact of this variant on the transcript level demonstrated partial aberrant splicing with residual expression of wildtype transcript. Thus, the molecular analyses provided a plausible explanation of the difference in clinical severity between the two cases and allowed the refinement of the diagnosis in the affected cats to JEB. This study highlights the complexity of EB in animals and contributes to a better understanding of the genotype-phenotype correlation in COL17A1-related JEB.
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Barbeau, Susie, Fannie Semprez, Alexandre Dobbertin, Laurine Merriadec, Florine Roussange, Bruno Eymard, Damien Sternberg et al. "Molecular Analysis of a Congenital Myasthenic Syndrome Due to a Pathogenic Variant Affecting the C-Terminus of ColQ". International Journal of Molecular Sciences 24, n. 22 (11 novembre 2023): 16217. http://dx.doi.org/10.3390/ijms242216217.
Abstract (sommario):
Congenital Myasthenic Syndromes (CMSs) are rare inherited diseases of the neuromuscular junction characterized by muscle weakness. CMSs with acetylcholinesterase deficiency are due to pathogenic variants in COLQ, a collagen that anchors the enzyme at the synapse. The two COLQ N-terminal domains have been characterized as being biochemical and functional. They are responsible for the structure of the protein in the triple helix and the association of COLQ with acetylcholinesterase. To deepen the analysis of the distal C-terminal peptide properties and understand the CMSs associated to pathogenic variants in this domain, we have analyzed the case of a 32 year old male patient bearing a homozygote splice site variant c.1281 C > T that changes the sequence of the last 28 aa in COLQ. Using COS cell and mouse muscle cell expression, we show that the COLQ variant does not impair the formation of the collagen triple helix in these cells, nor its association with acetylcholinesterase, and that the hetero-oligomers are secreted. However, the interaction of COLQ variant with LRP4, a signaling hub at the neuromuscular junction, is decreased by 44% as demonstrated by in vitro biochemical methods. In addition, an increase in all acetylcholine receptor subunit mRNA levels is observed in muscle cells derived from the patient iPSC. All these approaches point to pathophysiological mechanisms essentially characterized by a decrease in signaling and the presence of immature acetylcholine receptors.
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Tohar, Ran, Tamar Ansbacher, Inbal Sher, Livnat Afriat-Jurnou, Evgeny Weinberg e Maayan Gal. "Screening Collagenase Activity in Bacterial Lysate for Directed Enzyme Applications". International Journal of Molecular Sciences 22, n. 16 (9 agosto 2021): 8552. http://dx.doi.org/10.3390/ijms22168552.
Abstract (sommario):
Collagenases are essential enzymes capable of digesting triple-helical collagen under physiological conditions. These enzymes play a key role in diverse physiological and pathophysiological processes. Collagenases are used for diverse biotechnological applications, and it is thus of major interest to identify new enzyme variants with improved characteristics such as expression yield, stability, or activity. The engineering of new enzyme variants often relies on either rational protein design or directed enzyme evolution. The latter includes screening of a large randomized or semirational genetic library, both of which require an assay that enables the identification of improved variants. Moreover, the assay should be tailored for microplates to allow the screening of hundreds or thousands of clones. Herein, we repurposed the previously reported fluorogenic assay using 3,4-dihydroxyphenylacetic acid for the quantitation of collagen, and applied it in the detection of bacterial collagenase activity in bacterial lysates. This enabled the screening of hundreds of E. coli colonies expressing an error-prone library of collagenase G from C. histolyticum, in 96-well deep-well plates, by measuring activity directly in lysates with collagen. As a proof-of-concept, a single variant exhibiting higher activity than the starting-point enzyme was expressed, purified, and characterized biochemically and computationally. This showed the feasibility of this method to support medium-high throughput screening based on direct evaluation of collagenase activity.
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Townsend, Amanda Rose, Rebecca Asher, Timothy Jay Price, Chee Khoon Lee, Hilary Dorward, Val Gebski, Yoko Tomita, Niall C. Tebbutt e Jennifer Hardingham. "Single nucleotide polymorphisms (SNPs) in COL4A2, PPP1R17, and ARHGAPP44 and prognostic value in metastatic colorectal cancer (mCRC)." Journal of Clinical Oncology 36, n. 4_suppl (1 febbraio 2018): 720. http://dx.doi.org/10.1200/jco.2018.36.4_suppl.720.
Abstract (sommario):
720 Background: Our previous study has identified COL4A2, PPP1R17 and ARHGAP44 SNPs using whole exome sequencing with potential prognostic significance (Townsend et al. ASCO GI 2017). COL4A2 encodes a subunit of type IV collagen, the C-terminal of which is an inhibitor of angiogenesis. We assessed prognostic impact of these variants in patients from the phase III MAX study (capecitabine +/- bevacizumab (+/- mitomycin C)). An analysis of predictive effect on bevacizumab was also undertaken. Methods: DNA was extracted from archival macrodissected formalin fixed paraffin embedded tumor tissue and genotyped using Agena Bioscience MassARRAY system (AGRF). Univariate association of variant group (WT versus mutation (MT)) with progression free survival (PFS) and overall survival (OS) was assessed using Kaplan-Meier curves and Cox regression models. Logistic regression models were used to assess association with response rate (RR). A cox regression model with treatment, variant status and their interaction investigated if variants were predictive of bevacizumab effect. Results: Of the available 145 of 471 (31%) patients in the MAX study, 25 (17%) had COL4A2 MT, 29 (20%) PPP1R17 MT, 14 (10%) ARHGAP44 MT. Patient demographics were comparable across treatment groups and outcomes similar to whole study population. On univariate analysis median PFS was numerically longer for WT vs MT in all 3 variants, but these differences were not significant (COL4A2 WT 8.4m v MT 6.0m, p=0.09; PPP1R17 WT 7.8m v MT 7.5m, p=0.76; ARHGAP44 WT 8.2m v MT 6.5m, p=0.86). There was also no significant association between variant type and OS. Multivariate analysis for COL4A2 MT v WT showed no significant difference in PFS or OS (HR 1.42; 95% CI 0.91-2.22, p=0.13 and HR 1.33; 95% CI 0.85-2.1, p=0.21). There was no association between treatment response and variant status. Variant status was not predictive of bevazicumab efficacy for treatment response, PFS or OS. Conclusions: There was no significant prognostic or predictive impact of novel gene variants in patients treated with bevacizumab. This may be due to small numbers of MT variants in this study and further studies in larger populations may be useful.
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Stanbury, Katherine, Renata Stavinohova, Louise Pettitt, Chris Dixon, Ellen C. Schofield, Bryan Mclaughlin, Inka Pettinen et al. "Multiocular defect in the Old English Sheepdog: A canine form of Stickler syndrome type II associated with a missense variant in the collagen-type gene COL11A1". PLOS ONE 18, n. 12 (28 dicembre 2023): e0295851. http://dx.doi.org/10.1371/journal.pone.0295851.
Abstract (sommario):
Multiocular defect has been described in different canine breeds, including the Old English Sheepdog. Affected dogs typically present with multiple and various ocular abnormalities. We carried out whole genome sequencing on an Old English Sheepdog that had been diagnosed with hereditary cataracts at the age of five and then referred to a board-certified veterinary ophthalmologist due to owner-reported visual deterioration. An ophthalmic assessment revealed that there was bilateral vitreal degeneration, macrophthalmos, and spherophakia in addition to cataracts. Follow-up consultations revealed cataract progression, retinal detachment, uveitis and secondary glaucoma. Whole genome sequence filtered variants private to the case, shared with another Old English Sheepdog genome and predicted to be deleterious were genotyped in an initial cohort of six Old English Sheepdogs (three affected by multiocular defect and three control dogs without evidence of inherited eye disease). Only one of the twenty-two variants segregated correctly with multiocular defect. The variant is a single nucleotide substitution, located in the collagen-type gene COL11A1, c.1775T>C, that causes an amino acid change, p.Phe1592Ser. Genotyping of an additional 14 Old English Sheepdogs affected by multiocular defect revealed a dominant mode of inheritance with four cases heterozygous for the variant. Further genotyping of hereditary cataract-affected Old English Sheepdogs revealed segregation of the variant in eight out of nine dogs. In humans, variants in the COL11A1 gene are associated with Stickler syndrome type II, also dominantly inherited.
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Brown, J. C., K. Mann, H. Wiedemann e R. Timpl. "Structure and binding properties of collagen type XIV isolated from human placenta." Journal of Cell Biology 120, n. 2 (15 gennaio 1993): 557–67. http://dx.doi.org/10.1083/jcb.120.2.557.
Abstract (sommario):
Collagen XIV was isolated from neutral salt extracts of human placenta and purified by several chromatographic steps including affinity binding to heparin. The same procedures also led to the purification of a tissue form of fibronectin. Collagen XIV was demonstrated by partial sequence analysis of its Col1 and Col2 domains and by electron microscopy to be a disulphide-linked molecule with a characteristic cross-shape. The individual chains had a size of approximately 210 kD, which was reduced to approximately 180 kD (domain NC3) after treatment with bacterial collagenase. Specific antibodies mainly to NC3 epitopes were obtained by affinity chromatography and used in tissue and cell analyses by immunoblotting and radioimmunoassays. Two sequences from NC3 were identified on fragments obtained after trypsin cleavage. They were identical to cDNA-derived sequences of undulin, a noncollagenous extracellular matrix protein. This suggests that collagen XIV and undulin may be different splice variants from the same gene. Heparin binding was confirmed in ligand assays with a large basement membrane heparan sulphate proteoglycan. This binding could be inhibited by heparin and heparan sulphate but not by chondroitin sulphate. In addition, collagen XIV bound to the triple helical domain of collagen VI. The interactions with heparin sulphate proteoglycan and collagen VI were not shared by the NC3 domain, or by reduced and alkylated collagen XIV. No or only low binding was observed for collagens I-V, pN-collagens I and III, and several noncollagenous matrix proteins, including laminin, recombinant nidogen, BM-40/osteonectin, plasma and tissue fibronectin, vitronectin, and von Willebrand factor. Insignificant activity was also shown in cell attachment assays with nine established cell lines.
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Graves, Lara E., Christie-Lee Wall, Julie N. Briody, Bruce Bennetts, Karen Wong, Ella Onikul, Andrew Biggin e Craig F. Munns. "High Bone Mineral Density Osteogenesis Imperfecta in a Family with a Novel Pathogenic Variant in COL1A2". Hormone Research in Paediatrics 93, n. 4 (2020): 263–71. http://dx.doi.org/10.1159/000510463.
Abstract (sommario):
Osteogenesis imperfecta (OI) is a heterogenous group of heritable bone dysplasias characterized by bone fragility, typically low bone mass, joint laxity, easy bruising, and variable short stature. Classical OI is caused by autosomal dominant pathogenic variants in <i>COL1A1</i> or <i>COL1A2</i> that result in either reduced production of normal type 1 collagen or structurally abnormal collagen molecules. Pathogenic variants in these genes generally result in low bone mass. Here, we report a family that had 2 affected individuals who presented with minimal trauma fractures and were found to have elevated bone mineral density (BMD) and a previously unreported variant in <i>COL1A2</i> c.3356C>T p.(Ala1119Val). We report the change in BMD using dual-energy X-ray and peripheral quantitative computed tomography over a 2.3-year period in the proband. This case report highlights the importance of BMD studies and genetic testing in the diagnostic process for brittle bone disorders.
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Christen, Matthias, Henriëtte Booij-Vrieling, Jelena Oksa-Minalto, Cynthia de Vries, Alexandra Kehl, Vidhya Jagannathan e Tosso Leeb. "MIA3 Splice Defect in Cane Corso Dogs with Dental-Skeletal-Retinal Anomaly (DSRA)". Genes 12, n. 10 (25 settembre 2021): 1497. http://dx.doi.org/10.3390/genes12101497.
Abstract (sommario):
We investigated a hereditary syndrome in Cane Corso dogs. Affected dogs developed dental-skeletal-retinal anomaly (DSRA), clinically characterized by brittle, discolored, translucent teeth, disproportionate growth and progressive retinal degeneration resulting in vision loss. Combined linkage and homozygosity mapping delineated a 5.8 Mb critical interval. The comparison of whole genome sequence data of an affected dog to 789 control genomes revealed a private homozygous splice region variant in the critical interval. It affected the MIA3 gene encoding the MIA SH3 domain ER export factor 3, which has an essential role in the export of collagen and other secreted proteins. The identified variant, XM_005640835.3:c.3822+3_3822+4del, leads to skipping of two exons from the wild type transcript, XM_005640835.3:r.3712_3822del. Genotypes at the variant were consistent with monogenic autosomal recessive mode of inheritance in a complete family and showed perfect genotype-phenotype association in 18 affected and 22 unaffected Cane Corso dogs. MIA3 variants had previously been shown to cause related phenotypes in humans and mice. Our data in dogs together with the existing functional knowledge of MIA3 variants in other mammalian species suggest the MIA3 splice defect and a near complete loss of gene function as causative molecular pathomechanism for the DSRA phenotype in the investigated dogs.
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Bryant, Griffin, Peyton Moore e Mohanakrishnan Sathyamoorthy. "The Association of a Single Nucleotide Variant in COL5A1 to Early Onset Keratoconus and Pectus Excavatum—Convergence of Extracellular Matrix Pathologies". Medicina 60, n. 6 (13 giugno 2024): 974. http://dx.doi.org/10.3390/medicina60060974.
Abstract (sommario):
Keratoconus is a bilateral ocular condition characterized by irregularities and the thinning of the cornea. Decreased central corneal thickness is a hallmark of the condition, and numerous genes have played a role in altering corneal thickness and the subsequent development of keratoconus. Variants in the structural and regulatory genes of the extracellular matrix have been highly associated with keratoconus, as well as with pectus excavatum, a chest wall deformity commonly seen in connective tissue disorders. This report describes a patient with a c.1720-11T>A intronic variant in the collagen-encoding gene, COL5A1, who was diagnosed with early-onset keratoconus and demonstrated a significant pectus excavatum. This report associates a COL5A1 variant with these seemingly unrelated phenotypic associations, further advancing the literature on the topic.
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Daga, Sergio, Francesco Donati, Katia Capitani, Susanna Croci, Rossella Tita, Annarita Giliberti, Floriana Valentino et al. "New frontiers to cure Alport syndrome: COL4A3 and COL4A5 gene editing in podocyte-lineage cells". European Journal of Human Genetics 28, n. 4 (21 novembre 2019): 480–90. http://dx.doi.org/10.1038/s41431-019-0537-8.
Abstract (sommario):
AbstractAlport syndrome (AS) is an inherited genetic disorder characterized by range of alterations from glomerular basement membrane abnormalities up to end-stage renal disease. Pathogenic variants in the collagen α3, α4, and α5 encoding genes are causative both of the autosomal dominant and of the X-linked forms of AS. Podocytes are the only renal cells that are able to produce the COL(IV)a3-a4a5 heterotrimer. We have previously demonstrated how it is possible to isolate podocyte-lineage cells from urine of patients, providing an easily accessible cellular model closer to the podocytes’ physiological conditions. Taking advantage of disease-relevant cell lines, we employed a two-plasmid approach in order to achieve a beneficial and stable variant-specific correction using CRISPR/Cas9 genome editing. One plasmid carries a Donor DNA and a reporter system mCherry/GFP to track the activity of Cas9 in cells. The other plasmid carries a self-cleaving SpCas9 and the variant-specific sgRNA. We have analyzed two stable podocyte-lineage cell lines, harboring a variant in the X-linked COL4A5 (p.(Gly624Asp)) and in the autosomal COL4A3 gene (p.(Gly856Glu)). We have achieved reversion of variants greater than 40% with undesired insertions/deletions lower than 15%. Overall, we have demonstrated a new gene therapy approach directly on patients’ cells, key players of Alport pathogenesis, and we have reverted COL4 causative variants towards the wild type state. These results, in combination with preclinical models, could open new frontiers in the management and the treatment of the disorder.
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Tešanović Perković, Deša, Zrinka Bukvić Mokos e Branka Marinović. "Epidermolysis Bullosa Acquisita—Current and Emerging Treatments". Journal of Clinical Medicine 12, n. 3 (1 febbraio 2023): 1139. http://dx.doi.org/10.3390/jcm12031139.
Abstract (sommario):
Epidermolysis bullosa acquisita (EBA) is a rare chronic autoimmune subepidermal blistering disease of the skin and mucous membranes, usually beginning in adulthood. EBA is induced by autoantibodies to type VII collagen, a major component of anchoring fibrils in the dermal–epidermal junction (DEJ). The binding of autoantibodies to type-VII collagen subsequently leads to the detachment of the epidermis and the formation of mucocutaneous blisters. EBA has two major clinical subtypes: the mechanobullous and inflammatory variants. The classic mechanobullous variant presentation consists of skin fragility, bullae with minimal clinical or histological inflammation, erosions in acral distribution that heal with scarring, and milia formation. The inflammatory variant is challenging to differentiate from other autoimmune bullous diseases, most commonly bullous pemphigoid (BP) but also mucous membrane pemphigoid (MMP), Brunsting–Perry pemphigoid, and linear IgA dermatosis. Due to its recalcitrance conventional treatment of epidermolysis bullosa acquisita is shown to be demanding. Here we discuss novel therapeutic strategies that have emerged and which could potentially improve the quality of life in patients with EBA.
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Jacinto, Joana G. P., Irene M. Häfliger, Fintan J. McEvoy, Cord Drögemüller e Jørgen S. Agerholm. "A De Novo Mutation in COL1A1 in a Holstein Calf with Osteogenesis Imperfecta Type II". Animals 11, n. 2 (20 febbraio 2021): 561. http://dx.doi.org/10.3390/ani11020561.
Abstract (sommario):
Osteogenesis imperfecta (OI) type II is a genetic connective tissue disorder characterized by bone fragility, severe skeletal deformities and shortened limbs. OI usually causes perinatal death of affected individuals. OI type II diagnosis in humans is established by the identification of heterozygous mutations in genes coding for collagens. The purpose of this study was to characterize the pathological phenotype of an OI type II-affected neonatal Holstein calf and to identify the causative genetic variant by whole-genome sequencing (WGS). The calf had acute as well as intrauterine fractures, abnormally shaped long bones and localized arthrogryposis. Genetic analysis revealed a private heterozygous missense variant in COL1A1 (c.3917T>A) located in the fibrillar collagen NC1 domain (p.Val1306Glu) that most likely occurred de novo. This confirmed the diagnosis of OI type II and represents the first report of a pathogenic variant in the fibrillar collagen NC domain of COL1A1 associated to OI type II in domestic animals. Furthermore, this study highlights the utility of WGS-based precise diagnostics for understanding congenital disorders in cattle and the need for continued surveillance for rare lethal genetic disorders in cattle.
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Yousaf, Ahmed, Osama J. Boustany, Michael Gerbo, Shanawar Waris, Stephen Davis, Wei Fang e Roxann Powers. "Localized Versus Generalized Granuloma Annulare: A Retrospective Review of 407 Patients". Journal of Cutaneous Medicine and Surgery 25, n. 4 (24 febbraio 2021): 384–89. http://dx.doi.org/10.1177/1203475421996319.
Abstract (sommario):
Background Granuloma annulare has been linked to diabetes, dyslipidemia, thyroid disease, collagen vascular disease, malignancies, infectious hepatitis, and systemic infections. However, these associations have not been systematically investigated when categorized by its clinical variants. Objective To evaluate disease associations of localized and generalized granuloma annulare. Methods In total, 407 granuloma annulare patients from 1989 to 2019 were retrospectively reviewed, categorized by clinical variant (localized or generalized), age (pediatric or adult), and diagnostic method (clinical or histologic). Descriptive statistical analyses and multivariate logistic regression analysis were performed. Fisher’s exact tests were conducted to produce unbiased probability values. Results Overall, 75.2% of the study sample was female, 47.2% had dyslipidemia, 24.8% were diabetic, and 24.6% had thyroid disease. Dyslipidemia (OR 2.15, CI 1.95-2.35, P < .001), diabetes (OR 1.16, CI 1.01-1.31, P = .041), and histologic diagnosis (OR 2.08, CI 1.21-3.52, P = .007) were associated with increased risk of GGA compared to LGA. When stratified by adult versus pediatric cases, dyslipidemia and diagnostic method remained significant, but diabetes did not. Conclusions Evaluating granuloma annulare by its clinical variants may help to determine disease associations with each variant.
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Plachy, Lukas, Petra Dusatkova, Klara Maratova, Lenka Petruzelkova, Stanislava Kolouskova, Marta Snajderova, Barbora Obermannova et al. "Familial Short Stature - a Novel Phenotype of Growth Plate Collagenopathies". Journal of the Endocrine Society 5, Supplement_1 (1 maggio 2021): A723. http://dx.doi.org/10.1210/jendso/bvab048.1472.
Abstract (sommario):
Abstract Backround: Collagens are the most abundant proteins in the human body. In a growth plate, collagen types II, IX, X and XI are present. Defects in collagen genes cause heterogeneous syndromic disorders frequently associated with asymmetric short stature (e.g. Kniest dysplasia, spondyloepiphyseal dysplasia). Less is known about nonsyndromic collagenopathies - data about their frequency and subtle phenotypic signs are sparse, the information about their response to growth hormone (GH) treatment is lacking completely. Aim: To evaluate the frequency of collagenopathies in familial short stature (FSS) children and to describe their phenotype, including growth hormone (GH) treatment response. Methods: Out of 522 individuals treated in our center with GH from the indication of primary GH deficiency (GHD) or small for gestational age short stature (SGA-SS), 87 children with FSS fulfilled the inclusion criteria (pre-treatment height ≤-2 SD in both patient/their shorter parent, signed written informed consent) and were enrolled to the study. Next-generation sequencing was performed to search for variants in COL2A1, COL9A1, COL9A2, COL9A3, COL10A1, COL11A1 and COL11A2 genes. The results were evaluated using ACMG guidelines. The phenotype of children with (likely) pathogenic variants was described including the short-term GH treatment response (growth velocity and body-height SDS increase over three years of treatment). For statistical evaluation, parametric tests were used, p-values <0.05 were considered significant. Results: A (likely) pathogenic variant in one of the collagen genes was found in 10/87 (11.5%) children. Their age was 12.5 years (median, range 6-17 years), their pre-treatment height was -3.1 SD (-2.4 to -4.3 SD). Their birth length (median -2.8 SD; range -0.7 to -4.1 SD) was more severely affected than birth weight (median -2.1 SD; range -1.0 to -2.7 SD). Eight children were treated with GH from SGA-SS indication, the remaining 2 were classified as mild GHD (maximal stimulated GH concentration 8.0 and 9.7 ug/l, normal brain MRI and examination of other pituitary hormones). Detailed anthropometric examination described mild asymmetry with shorter limbs and mild bone dysplasia signs (scoliosis, more pronounced lumbar lordosis, genua valga, limited elbow extension) in 2/10 and 4/10 affected children, respectively. Growth velocity improved from a median of 5.3 cm/year to 8.7 cm/year after one year of treatment (p<0.001, paired-sample T-test), height improved from a median of -3.1 SD to -2.2 SD after three years of therapy (p=0.001, ANOVA repeated measures analysis of variants). Conclusion: Nonsyndromic collagenopathies are a frequent cause of FSS. The short-term response to GH treatment is promising. Supported by the Ministry of Health, Czech Republic, grant number NV18- 07-00283 and by the research project of the Grant Agency of Charles University of Prague, GAUK 976718.
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Saqlain, Muhammad, Madiha Khalid, Muhammad Fiaz, Sadia Saeed, Asad Mehmood Raja, Muhammad Mobeen Zafar, Tahzeeb Fatima et al. "Risk variants of obesity associated genes demonstrate BMI raising effect in a large cohort". PLOS ONE 17, n. 9 (20 settembre 2022): e0274904. http://dx.doi.org/10.1371/journal.pone.0274904.
Abstract (sommario):
Obesity is highly polygenic disease where several genetic variants have been reportedly associated with obesity in different ethnicities of the world. In the current study, we identified the obesity risk or protective association and BMI raising effect of the minor allele of adiponectin, C1Q and collagen domain containing (ADIPOQ), cholesteryl ester transfer protein (CEPT), FTO alpha-ketoglutarate dependent dioxygenase (FTO), leptin (LEP), and leptin receptor (LEPR) genes in a large cohort stratified into four BMI-based body weight categories i.e., normal weight, lean, over-weight, and obese. Based on selected candidate genetic markers, the genotyping of all study subjects was performed by PCR assays, and genotypes and allele frequencies were calculated. The minor allele frequencies (MAFs) of all genetic markers were computed for total and BMI-based body weight categories and compared with MAFs of global and South Asian (SAS) populations. Genetic associations of variants with obesity risk were calculated and BMI raising effect per copy of the minor allele were estimated. The genetic variants with higher MAFs in obese BMI group were; rs2241766 (G = 0.43), rs17817449 (G = 0.54), rs9939609 (A = 0.51), rs1421085 (C = 0.53), rs1558902 (A = 0.63), and rs1137101 (G = 0.64) respectively. All these variants were significantly associated with obesity (OR = 1.03–4.42) and showed a high BMI raising effect (β = 0.239–0.31 Kg/m2) per copy of the risk allele. In contrast, the MAFs of three variants were higher in lean-normal BMI groups; rs3764261 A = 0.38, rs9941349 T = 0.43, and rs7799039 G = 0.40–0.43). These variants showed obesity protective associations (OR = 0.68–0.76), and a BMI lowering effect per copy of the protective allele (β = -0.103–0.155 Kg/m2). The rs3764261 variant also showed significant and positive association with lean body mass (OR = 2.38, CI = 1.30–4.34). Overall, we report six genetic variants of ADIPOQ, FTO and LEPR genes as obesity-risk markers and a CETP gene variant as lean mass/obesity protective marker in studied Pakistani cohort.
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Storoni, Silvia, Sara J. E. Verdonk, Lidiia Zhytnik, Gerard Pals, Sanne Treurniet, Mariet W. Elting, Ralph J. B. Sakkers, Joost G. van den Aardweg, Elisabeth M. W. Eekhoff e Dimitra Micha. "From Genetics to Clinical Implications: A Study of 675 Dutch Osteogenesis Imperfecta Patients". Biomolecules 13, n. 2 (2 febbraio 2023): 281. http://dx.doi.org/10.3390/biom13020281.
Abstract (sommario):
Osteogenesis imperfecta (OI) is a heritable connective tissue disorder that causes bone fragility due to pathogenic variants in genes responsible for the synthesis of type I collagen. Efforts to classify the high clinical variability in OI led to the Sillence classification. However, this classification only partially takes into account extraskeletal manifestations and the high genetic variability. Little is known about the relation between genetic variants and phenotype as of yet. The aim of the study was to create a clinically relevant genetic stratification of a cohort of 675 Dutch OI patients based on their pathogenic variant types and to provide an overview of their respective medical care demands. The clinical records of 675 OI patients were extracted from the Amsterdam UMC Genome Database and matched with the records from Statistics Netherlands (CBS). The patients were categorized based on their harbored pathogenic variant. The information on hospital admissions, outpatient clinic visits, medication, and diagnosis-treatment combinations (DTCs) was compared between the variant groups. OI patients in the Netherlands appear to have a higher number of DTCs, outpatient clinic visits, and hospital admissions when compared to the general Dutch population. Furthermore, medication usage seems higher in the OI cohort in comparison to the general population. The patients with a COL1A1 or COL1A2 dominant negative missense non-glycine substitution appear to have a lower health care need compared to the other groups, and even lower than patients with COL1A1 or COL1A2 haploinsufficiency. It would be useful to include the variant type in addition to the Sillence classification when categorizing a patient’s phenotype.
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Kurihara, Hiroki, Yasushi Imai, Takao Sugiyama, Chikuma Hamada, Eiichi Sakai, Mitsuko Mori, Ryozo Nagai e Hiroyuki Morita. "Lack of Association between the Platelet Glycoprotein Ia C807T Gene Polymorphism and Myocardial Infarction in Japanese". Thrombosis and Haemostasis 85, n. 02 (2001): 226–30. http://dx.doi.org/10.1055/s-0037-1615702.
Abstract (sommario):
SummaryThe platelet-collagen receptor, glycoprotein Ia/IIa (integrin α2β1) plays a fundamental role in the adhesion of platelets to fibrillar collagen, an event leading to platelet activation and thrombus formation and contributing to the pathogenesis of thrombotic disease. Further, glyco-protein Ia/IIa receptor density and function may be associated with two linked and silent polymorphisms (807C/T and 873G/A) within the glyco-protein Ia gene. We tested the extent to which these polymorphisms serve as genetic markers of myocardial infarction in a Japanese population. A case-control study was carried out using 210 Japanese myocar-dial infarction patients and 420 age- and sex-matched controls. Geno-typing was accomplished using PCR followed by melting curve analysis with specific fluorescent hybridization probes. The 807CC, CT, TT genotypes linked perfectly to the 873GG, GA, AA genotypes, respectively. Allele frequencies of the 807T (873A) variant were similar in the control and patient groups (0.373 vs. 0.352). The 807T and 873A variants of platelet glycoprotein Ia gene are common and in a perfect linkage in the Japanese population, but it appears unlikely that the 807T (873A) variant represents a useful marker of increased risk for myocardial infarction.
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Koshevaya, Yulia S., Mariia E. Turkunova, Anastasia O. Vechkasova, Elena A. Serebryakova, Maxim Yu Donnikov, Svyatoslav I. Papanov, Alexander N. Chernov et al. "Exome Sequencing for the Diagnostics of Osteogenesis Imperfecta in Six Russian Patients". Current Issues in Molecular Biology 46, n. 5 (29 aprile 2024): 4106–18. http://dx.doi.org/10.3390/cimb46050252.
Abstract (sommario):
Osteogenesis imperfecta (OI) is a group of inherited disorders of connective tissue that cause significant deformities and fragility in bones. Most cases of OI are associated with pathogenic variants in collagen type I genes and are characterized by pronounced polymorphisms in clinical manifestations and the absence of clear phenotype–genotype correlation. The objective of this study was to conduct a comprehensive molecular–genetic and clinical analysis to verify the diagnosis of OI in six Russian patients with genetic variants in the COL1A1 and COL1A2 genes. Clinical and laboratory data were obtained from six OI patients who were observed at the Medical Genetics Center in Saint Petersburg from 2016 to 2023. Next-generation sequencing on MGISEQ G400 (MGI, China) was used for DNA analysis. The GATK bioinformatic software (version 4.5.0.0) was used for variant calling and hard filtering. Genetic variants were verified by the direct automatic sequencing of PCR products using the ABI 3500X sequencer. We identified six genetic variants, as follows pathogenic c.3505G>A (p. Gly1169Ser), c.769G>A (p.Gly257Arg), VUS c.4123G>A (p.Ala1375Thr), and c.4114A>T (p.Asn1372Tyr) in COL1A1; and likely pathogenic c.2035G>A (p.Gly679Ser) and c.739-2A>T in COL1A2. In addition, clinical cases are presented due to the presence of the c.4114A>T variant in the COL1A2 gene. Molecular genetics is essential for determining different OI types due to the high similarity across various types of the disease and the failure of unambiguous diagnosis based on clinical manifestations alone. Considering the variable approaches to OI classification, an integrated strategy is required for optimal patient management.
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Nixon, Thomas R. W., Allan J. Richards, Howard Martin, Philip Alexander e Martin P. Snead. "Autosomal Recessive Stickler Syndrome". Genes 13, n. 7 (24 giugno 2022): 1135. http://dx.doi.org/10.3390/genes13071135.
Abstract (sommario):
Stickler syndrome (SS) is a genetic disorder with manifestations in the eye, ear, joints, face and palate. Usually inherited in a dominant fashion due to heterozygous pathogenic variants in the collagen genes COL2A1 and COL11A1, it can rarely be inherited in a recessive fashion from variants in COL9A1, COL9A2, and COL9A3, COL11A1, as well as the non-collagen genes LRP2, LOXL3 and GZF1. We review the published cases of recessive SS, which comprise 40 patients from 23 families. Both homozygous and compound heterozygous pathogenic variants are found. High myopia is near-universal, and sensorineural hearing loss is very common in patients with variants in genes for type IX or XI collagen, although hearing appears spared in the LRP2 and LOXL3 patients and is variable in GZF1. Cleft palate is associated with type XI collagen variants, as well as the non-collagen genes, but is so far unreported with type IX collagen variants. Retinal detachment has occurred in 18% of all cases, and joint pain in 15%. However, the mean age of this cohort is 11 years old, so the lifetime incidence of both problems may be underestimated. This paper reinforces the importance of screening for SS in congenital sensorineural hearing loss, particularly when associated with myopia, and the need to warn patients and parents of the warning signs of retinal detachment, with regular ophthalmic review.
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Chiarelli, Ritelli, Zoppi e Colombi. "Cellular and Molecular Mechanisms in the Pathogenesis of Classical, Vascular, and Hypermobile Ehlers‒Danlos Syndromes". Genes 10, n. 8 (12 agosto 2019): 609. http://dx.doi.org/10.3390/genes10080609.
Abstract (sommario):
The Ehlers‒Danlos syndromes (EDS) constitute a heterogenous group of connective tissue disorders characterized by joint hypermobility, skin abnormalities, and vascular fragility. The latest nosology recognizes 13 types caused by pathogenic variants in genes encoding collagens and other molecules involved in collagen processing and extracellular matrix (ECM) biology. Classical (cEDS), vascular (vEDS), and hypermobile (hEDS) EDS are the most frequent types. cEDS and vEDS are caused respectively by defects in collagen V and collagen III, whereas the molecular basis of hEDS is unknown. For these disorders, the molecular pathology remains poorly studied. Herein, we review, expand, and compare our previous transcriptome and protein studies on dermal fibroblasts from cEDS, vEDS, and hEDS patients, offering insights and perspectives in their molecular mechanisms. These cells, though sharing a pathological ECM remodeling, show differences in the underlying pathomechanisms. In cEDS and vEDS fibroblasts, key processes such as collagen biosynthesis/processing, protein folding quality control, endoplasmic reticulum homeostasis, autophagy, and wound healing are perturbed. In hEDS cells, gene expression changes related to cell-matrix interactions, inflammatory/pain responses, and acquisition of an in vitro pro-inflammatory myofibroblast-like phenotype may contribute to the complex pathogenesis of the disorder. Finally, emerging findings from miRNA profiling of hEDS fibroblasts are discussed to add some novel biological aspects about hEDS etiopathogenesis.
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Agardh, Elisabeth, Gayle Teramura, Prashart Gaur, Lakshmi Gaur, Carl-David Agardh e Alexander Reiner. "Diabetes duration may modify the association between genetic variation in the glycoprotein Ia subunit of the platelet collagen receptor and risk of severe diabetic retinopathy: a working hypothesis". Thrombosis and Haemostasis 89, n. 01 (2003): 142–48. http://dx.doi.org/10.1055/s-0037-1613553.
Abstract (sommario):
SummaryGenetic factors appear to contribute to the severity and progression of diabetic retinopathy. We assessed the associations of the C807T and Glu505Lys variants of the glycoprotein Ia ( 2 integrin) subunit of the platelet/endothelial collagen receptor and risk of retinopathy in a population-based survey of 288 diabetic patients in one Swedish community. Neither variant was associated with retinopathy risk overall. However, the 807T variant was associated with increased risk of severe retinopathy, and the association was modified by diabetes duration. Among patients with diabetes of longer duration ( 25 years), the 807T variant was strongly associated with risk of severe retinopathy (odds ratio 7.49, 95% confidence interval 1.75 to 32.1). There was no association between the 807T variant and risk of severe retinopathy among patients with diabetes duration <25 years. The Lys505 variant of glycoprotein Ia was associated with an odds ratio for severe retinopathy of 1.88 (95% confidence interval 0.83 to 4.24). Overall, there was a significant interaction between glycoprotein Ia genotype and duration of diabetes on the risk of retinopathy (P-value for interaction = 0.019).These results suggest the hypothesis that genetic variation of platelet glycoprotein Ia may play a particularly important role during the advanced stages of diabetic retinopathy.
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Kilianova, Zuzana, Natalia Ciznarova, Kristina Szmicsekova, Lubica Slobodova e Anna Hrabovska. "Expression of cholinesterases and their anchoring proteins in rat heart". Canadian Journal of Physiology and Pharmacology 98, n. 7 (luglio 2020): 473–76. http://dx.doi.org/10.1139/cjpp-2019-0565.
Abstract (sommario):
Acetylcholine (ACh)-mediated vagal transmission as well as nonneuronal ACh release are considered cardioprotective in pathological situations with increased sympathetic drive such as ischemia–reperfusion and cardiac remodeling. ACh action is terminated by hydrolysis by the cholinesterases (ChEs), acetylcholinesterase, and butyrylcholinesterase. Both ChEs exist in multiple molecular variants either soluble or anchored by specific anchoring proteins like collagen Q (ColQ) anchoring protein and proline-rich membrane anchoring protein (PRiMA). Here we assessed the expression of specific ChE molecular forms in different heart compartments using RT-qPCR. We show that both ChEs are expressed in all heart compartments but display different expression patterns. The acetylcholinesterase-T variant together with PRiMA and ColQ is predominantly expressed in rat atria. Butylcholinesterase is found in all heart compartments and is accompanied by both PRiMA and ColQ anchors. Its expression in the ventricular system suggests involvement in the nonneuronal cholinergic system. Additionally, two PRiMA variants are detected throughout the rat heart.
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Brake, Marisa A., Audrey C. Cleuren, Dakota R. Redshaw, Caitlin Schneider, Aaron Scholl, Adrianna Jurek, Martijn A. van der Ent e Randy J. Westrick. "An Intergenic Noncoding Chromosome 18 Variant Suppresses Lethal Thrombosis in Mice By Normalizing Blood Coagulation and Reducing Platelet Reactivity". Blood 138, Supplement 1 (5 novembre 2021): 442. http://dx.doi.org/10.1182/blood-2021-153531.
Abstract (sommario):
Abstract Background: Factor V Leiden (FVL) is a common thrombosis susceptibility variant in humans. It is incompletely penetrant; this indicates that there are modifiers of FVL that alter thrombosis susceptibility. We used mouse models of FVL (F5L) and heterozygous tissue factor pathway inhibitor deficiency (Tfpi+/-), to identify a perinatal lethal genetic interaction when mice inherited F5L/L Tfpi+/-. This phenotype was used as the basis for a sensitized genome wide ENU mutagenesis screen to identify mutations suppressing lethal thrombosis in F5L/L Tfpi+/- mice. From this screen, we generated multiple independent lines of thrombosuppressed mice, called MF5L, for Modifier of F5L. MF5L16 was a large, highly penetrant (77.2%), multigenerational pedigree containing 136 viable F5L/L Tfpi+/- mice. Aims: In the present study, we aimed to identify and functionally characterize the thrombosuppressor mutation present in MF5L16. Methods: Genomic analyses: We performed whole genome sequencing (WGS) on four MF5L16 F5L/L Tfpi+/- mice. We used comparative bioinformatic analyses to identify variants inherited by all four mice and compiled these variants into candidate variant list. PCR and Sanger sequencing were used to analyze the 136 F5L/L Tfpi+/- mice for inheritance of each of the candidate variants. Functional analyses: We performed biochemical blood coagulation and platelet assays of blood from the Chr18 A mice . Complete blood counts were measured using the Advia 2120 with settings optimized for C57BL/6 mouse blood. Platelet aggregation studies were performed using the Roche Multiplate Aggregometer with ADP and type 1 collagen as the aggregating agents. Results: We analyzed four MF5L16 mice by WGS and identified seven spontaneous mutations that arose in our F5L/L breeding colony that were introduced into MF5L16. Importantly, no coding variants were linked to these variants. Analysis of these seven mutations in all 136 MF5L16 F5L/L Tfpi+/- mice revealed a significant association between a Chromosome 18 intergenic variant (Chr18 G to A, Chr18 A) and F5L/L Tfpi+/- mouse survival (p=0.003). To re-create the suppression of the lethal F5L/L Tfpi+/- phenotype, we bred F5+/L Tfpi+/- Chr18 +/A triple heterozygous mice to F5L/L Chr18 A/A mice to observe the effects of Chr18 A on F5L/L Tfpi+/- mouse survival. Out of 109 mice from this cross, two F5L/L Tfpi+/- Chr18 +/A mice were produced (expected ratio ~1:8). This suggests that the Chr18 A variant suppresses F5L/L Tfpi+/- lethal thrombosis at ~15% penetrance. Complete blood count analysis on Chr18 +/+,Chr18 +/A, and Chr18 A/A mice determined that Chr18 A/A mice had reduced platelet count and distribution width and increased variability in red blood cell (RBC) mean corpuscular volume (n≥4; p<0.05). The Chr18 A/A mice did not display differences in PT or aPTT assays, but had significantly reduced platelet aggregation velocity when stimulated by both ADP and collagen agonists (n≥4; p=0.0002). Additionally, blood smears revealed the presence of poikilocytic RBCs in the Chr18 A/A mice. Conclusions and future directions: Our results establish that a noncoding intergenic Chr18 variant at nucleotide position 62,970,011 (G>A, Chr18 A) contributes to thrombosuppression by reducing platelet reactivity. The observed platelet and RBC phenotypes suggest that a major mechanism of Chr18 A thrombosuppression could be through regulation of gene expression in cells of the myeloid lineage. We are performing additional platelet and blood coagulation analyses to refine the phenotypic differences due to the Chr18 A variant. Comparative transcriptomic analyses are also being performed to identify the genetic pathways involved. Understanding the mechanism in which this intergenic mutation suppresses thrombosis could provide insights into human thrombosis regulation. Disclosures No relevant conflicts of interest to declare.
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Snodgrass, James L., Nehal Mohamed, Julia M. Ross, Subrata Sau, Chia Y. Lee e Mark S. Smeltzer. "Functional Analysis of the Staphylococcus aureus Collagen Adhesin B Domain". Infection and Immunity 67, n. 8 (1 agosto 1999): 3952–59. http://dx.doi.org/10.1128/iai.67.8.3952-3959.1999.
Abstract (sommario):
ABSTRACT The Staphylococcus aureus collagen adhesin (CNA) occurs in at least four forms that differ in the number (one, two, three, or four) of B domains. The B domains contain 187 amino acids and are located between the domains that anchor CNA to the cell envelope and the ligand-binding A domain. To determine whether a B domain is required for functional expression of CNA, we cloned the 2Bcna gene from S. aureus strain Phillips and then eliminated both B domains by overlapping PCR. The absence of a B domain did not affect processing of the collagen adhesin to the cell surface or the ability to bind collagen. Based on our recent demonstration that the capsule can mask CNA on the surface of S. aureus cells (A. F. Gillaspy et al., Infect. Immun. 66:3170–3178, 1998), we also investigated the possibility that multiple B domains can extend the ligand-binding A domain outward from the cell surface and thereby overcome the inhibitory effect of the capsule. Specifically, we cloned the naturally occurring 4B CNA variant from S. aureus UAMS-639 and, by successive elimination of B domains, generated 1, 2, and 3B variants that are isogenic with respect to the 4B clone. After introducing each variant into microencapsulated and heavily encapsulated strains of S. aureus and growing cells under conditions known to affect capsule production (e.g., growth on Columbia agar), we correlated capsule production with exposure of CNA on the cell surface and the ability to bind collagen. Under no circumstance was the masking effect of the capsule reduced by the presence of multiple B domains. These results indicate that the B domains do not extend the ligand-binding A domain outward in a fashion that can overcome the inhibition of collagen binding associated with capsule production.
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McKinnon, Thomas A. J., Agata Anna Nowak, Alina Hua, Carolyn Millar e Michael Laffan. "Flow Analysis of Von Willebrand Factor Collagen Binding Mutants". Blood 118, n. 21 (18 novembre 2011): 2213. http://dx.doi.org/10.1182/blood.v118.21.2213.2213.
Abstract (sommario):
Abstract Abstract 2213 Von Willebrand Factor (VWF) binds to exposed sub-endothelial collagen at sites of vessel injury principally via its A3 domain, although some evidence suggests that the A1 domain can compensate for the A3 domain under flow conditions if the A3 domain is absent or non-functional. Recently, several naturally occurring Von Willebrand disease-causing mutations have been indentified in the A3 domain; S1731T, W1745C, S1783, H1786D and most recently M1761K, as well as one mutation in the A1 domain (I1343V) all of which have defective collagen binding. While the collagen binding function of these mutations has been assessed under static conditions it remains to be established if these affect collagen binding under shear stress. In the present study the collagen binding mutants were expressed in HEK293T cells and collagen binding function determined using an in vitro flow assay. All of the mutations were expressed at similar levels to wild type (wt) VWF and demonstrated normal multimeric patterns and binding to GPIbα under static conditions. As expected, collagen binding analysis under static conditions confirmed the collagen binding defect of all the mutants, with reduced or abolished binding to both collagens type I and III for all the mutants except S1731T which demonstrated normal binding to collagen type III and slightly reduced binding to collagen type I. Analysis of platelet capture under flow conditions confirmed that all the mutants were able to capture platelets similarly to wtVWF. Analysis of VWF mediated platelet capture to a collagen surface under flow conditions confirmed the phenotype of the collagen binding mutants. With the exception of S1731T, which demonstrated normal platelet capture on both collagens, none of the mutants were able to bind to collagen type I or III under flow conditions, or mediate platelet capture at high shear stress. The collagen binding function of these mutants under flow was partially restored when co-expressed with wtVWF. Interestingly, in contrast to a previous study, a VWF variant lacking the A3 domain (VWF-ΔA3) failed to bind to collagen under shear stress and was not able to mediate platelet capture to collagen. Together these data confirm that the major collagen binding site in VWF is located in the A3 domain and demonstrate that collagen binding mutations affect VWF mediated platelet capture under shear stress. Disclosures: No relevant conflicts of interest to declare.
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Garcia, Teresa Maria, Sarah Kiener, Vidhya Jagannathan, Duncan S. Russell e Tosso Leeb. "A COL7A1 Variant in a Litter of Neonatal Basset Hounds with Dystrophic Epidermolysis Bullosa". Genes 11, n. 12 (4 dicembre 2020): 1458. http://dx.doi.org/10.3390/genes11121458.
Abstract (sommario):
We investigated three neonatal Basset Hound littermates with lesions consistent with epidermolysis bullosa (EB), a group of genetic blistering diseases. A clinically normal bitch was bred to her grandfather by artificial insemination. Out of a litter of seven puppies, two affected puppies died and one was euthanized, with these puppies being submitted for diagnostic necropsy. All had multiple bullae and ulcers involving the nasal planum and paw pads, as well as sloughing claws; one puppy also had oral and esophageal ulcers. The complete genome of one affected puppy was sequenced, and 37 known EB candidate genes were assessed. We found a candidate causative variant in COL7A1, which encodes the collagen VII alpha 1 chain. The variant is a complex rearrangement involving duplication of a 107 bp region harboring a frameshift deletion of 7 bp. The variant is predicted to truncate more than 75% of the open reading frame, p.(Val677Serfs*11). Targeted genotyping of this duplication confirmed that all three affected puppies were homozygous for the duplication, whereas 12 unaffected Basset Hounds did not carry the duplication. This variant was also not seen in the genomes of more than 600 dogs of other breeds. COL7A1 variants have been identified in humans and dogs with dystrophic epidermolysis bullosa (DEB). The identified COL7A1 variant therefore most likely represents the causative variant and allows the refinement of the preliminary EB diagnosis to DEB.
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Zadro, Renata, Dunja Rogić, Désirée Coen-Herak, Ernest Bilić, Sara Dejanović Bekić, Margareta Radić Antolic e Ivana Lapić. "Reevaluation of von Willebrand disease diagnosis in a Croatian paediatric cohort combining bleeding scores, phenotypic laboratory assays and next generation sequencing". Biochemia medica 32, n. 1 (15 febbraio 2022): 85–97. http://dx.doi.org/10.11613/bm.2022.010707.
Abstract (sommario):
This study reevaluated von Willebrand disease (vWD) diagnosis in a Croatian paediatric cohort by combining bleeding scores (BS), phenotypic laboratory testing, and next-generation sequencing (NGS). A total of 25 children (11 males and 14 females, median age 10 years, from 2 to 17) previously diagnosed with vWD were included. BS were calculated using an online bleeding assessment tool. Phenotypic laboratory analyses included platelet count, platelet function analyser closure times, prothrombin time, activated partial thromboplastin time, von Willebrand factor antigen (vWF:Ag), vWF gain-of-function mutant glycoprotein Ib binding activity (vWF:GPIbM), vWF collagen binding activity (vWF:CBA), factor VIII activity (FVIII:C) and multimeric analysis. Next-generation sequencing covered regions of both vWF and FVIII genes and was performed on MiSeq (Illumina, San Diego, USA). Disease-associated variants identified in 15 patients comprised 11 distinct heterozygous vWF gene variants in 13 patients and one novel FVIII gene variant (p.Glu2085Lys) in two male siblings. Four vWF variants were novel (p.Gln499Pro, p.Asp1277Tyr, p.Asp1277His, p.Lys1491Glu). Three patients without distinctive variants had vWF:GPIbM between 30 and 50%. Patients with identified vWF gene variants had statistically significant lower values of vWF:GPIbM (P = 0.002), vWF:Ag (P = 0.007), vWF:CBA (P < 0.001) and FVIII:C (P = 0.002), compared to those without. Correlations between BS and phenotypic laboratory test results were not statistically significant for either of the tests. The applied diagnostic approach confirmed the diagnosis of vWD in 13 patients and mild haemophilia A in two. Limited utility of BS in the paediatric population was evidenced.
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Aksenova, M. E., P. E. Povilaitite, N. E. Konkova e V. V. Dlin. "Diagnostic Value of Type IV Collagen Expression in Renal Glomeruli at Alport’s Syndrome". Rossiyskiy Vestnik Perinatologii i Pediatrii (Russian Bulletin of Perinatology and Pediatrics) 65, n. 6 (22 gennaio 2021): 42–49. http://dx.doi.org/10.21508/1027-4065-2020-65-6-42-49.
Abstract (sommario):
The Alport’s syndrome is the hereditary multisystem disease characterized by the development of the progressive nephropathy. The early diagnosis and subsequent prescription of nephroprotective therapy improves significantly the nephrological prognosis. Purpose of the Study. Determine the value of the immunohistochemical method for the Alport’s syndrome diagnosis. Material and methods. The clinical, laboratory and morphological data of 35 patients with suspected Alport’s syndrome (13 years of age [11; 16]; 18 boys and 17 girls) examined in the Nephrology Department in 2013–2019 were summarized. The study of the renal tissue included the light, immunofluorescence, electron microscopy of the kidney biopsy sample, determination of the expression of α1, α3 and α5 chains of type IV collagen in the renal glomeruli using the immunohistochemical method; the genetic testing was carried out for 26 patients. The children were divided into groups depending on the glomerular expression of α5 chain of type IV collagen: normal (group 1, n=18), decreased (group 2, n=4), negative (group 3, n=13). Results are as the following: The disorder of the expression of α5 chain was detected in ¾ (q = 0.78) patients with genetically confirmed Alport’s syndrome and in almost all children with the X-linked variant of the disease (q = 0.94). Results. Based on the genetic testing, the Alport’s syndrome was confirmed in ¼ of the children of the 1st group (the children with the heterozygous variants of COL4A3, COL4A5 genes) and in all children of the 2nd and 3rd groups (COL4A5 variants). The sensitivity/ specificity of the immunohistochemical study for the Alport’s syndrome diagnosis was 78% /100%, that of the electron microscopy – 93% /87%. The predictive value of the positive/negative result of the immunohistochemical study was 100% /66%, that of the electron microscopy – 95% / 88% compared with 100% / 88% with the combine use of two methods. Conclusion. The determination of the expression of α5 chain of type IV collagen in the renal glomeruli has the independent diagnostic value, but it is inferior to the electron microscopy in the heterozygous variants of the Alport’s syndrome. The high specificity of the immunohistochemical method makes it possible to confirm the Alport’s syndrome in the case of the change in the expression of α5 chain of type IV collagen in the renal glomeruli.
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Teige, B., G. Gogstad, F. Brosstad e B. Olaisen. "Common structural genes for platelet and plasma fibrinogen". Blood 65, n. 1 (1 gennaio 1985): 120–26. http://dx.doi.org/10.1182/blood.v65.1.120.120.
Abstract (sommario):
Abstract Fibrinogen from plasma was compared with fibrinogen from platelets using two-dimensional electrophoresis. The source of platelet fibrinogen was isolated alpha-granules, thrombin- and collagen-released platelet material. The B beta- and gamma-chains from the different sources showed similar two-dimensional patterns, while gamma'-chains were not observed in platelet fibrinogen preparations. Furthermore, the A alpha-chain could hardly be identified in platelet preparations. When individual fibrinogen was studied in persons heterozygous for genetic B beta- and gamma-chain variants, the two-dimensional variant pattern could be demonstrated in plasma fibrinogen as well as in platelet fibrinogen. This observation strongly indicates that the structural genes for plasma and platelet fibrinogen B beta- and gamma-chains are identical.
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Teige, B., G. Gogstad, F. Brosstad e B. Olaisen. "Common structural genes for platelet and plasma fibrinogen". Blood 65, n. 1 (1 gennaio 1985): 120–26. http://dx.doi.org/10.1182/blood.v65.1.120.bloodjournal651120.
Abstract (sommario):
Fibrinogen from plasma was compared with fibrinogen from platelets using two-dimensional electrophoresis. The source of platelet fibrinogen was isolated alpha-granules, thrombin- and collagen-released platelet material. The B beta- and gamma-chains from the different sources showed similar two-dimensional patterns, while gamma'-chains were not observed in platelet fibrinogen preparations. Furthermore, the A alpha-chain could hardly be identified in platelet preparations. When individual fibrinogen was studied in persons heterozygous for genetic B beta- and gamma-chain variants, the two-dimensional variant pattern could be demonstrated in plasma fibrinogen as well as in platelet fibrinogen. This observation strongly indicates that the structural genes for plasma and platelet fibrinogen B beta- and gamma-chains are identical.