Knobloch syndrome in a patient from Chile.

Knobloch Syndrome (KS) is a rare autosomal recessive hereditary disease. Despite its clinical heterogeneity, it is characterized by vitreoretinal degeneration and high myopia, with or without occipital skull defects. It is caused by mutations in the COL18A1 gene, which codifies for collagen XVIII, present in retina and vascular endothelium. Since the first description of the disease by doctors Knobloch and Layer in 1972, over 100 cases and 20 pathogenic or likely pathogenic mutations have been reported. We present the case of a child born from a consanguineous couple in Chile with congenital high myopia and dysmorphisms without an occipital skull defect. Whole exome sequencing analysis revealed an inherited homozygous variant in COL18A1, c.4224_4225delinsC, p.Pro1411Leufs*35.

Collagen XVIII and LOXL-4 polymorphisms in women with and without advanced pelvic organ prolapse.

INTRODUCTION AND HYPOTHESIS: We verified the presence of single nucleotide polymorphisms (SNP) rs2236479 of the collagen 18 (COL18A1) and rs2862296 of the lysyl oxidase-like 4 (LOXL-4) genes and the association with pelvic organ prolapse (POP) in Brazilian women and determined risk factors for POP development. METHODS: We assessed 532 postmenopausal women divided into POP (stages III and IV) and control (stages 0 and I) groups by examination and peripheral blood sample collection. DNA sequences of interest were analyzed by real-time reverse-transcriptase polymerase chain reaction (RT-PCR). We used logistic regression models for the analyses, with p < 0.005 for significance. RESULTS: The frequency of homozygous polymorphic alleles (AA) in COL18A1 and (GG) in LOXL-4 were similar in both groups (17.5% and 15.4% for COL18A1 and 18.9% and 20.6% for LOXL-4, respectively). There were no associations between those polymorphisms or other genotypes and POP. Multiple logistic regression analysis identified age [odds ratio (OR) = 1.10, confidence interval (CI) 95% = 1.07; 1.14), number of vaginal births (OR = 1.66, CI 95% = 1.36; 2.03), and family history (OR = 2.55 CI 95% = 1.43; 4.55) as independent risk factors for POP. CONCLUSION: Our study suggests lack of association between DNA polymorphisms rs2236479 of COL18A1 and rs2862296 of LOXL-4 with advanced POP in this population.

Vascular endothelial growth factor as a biomarker for endostatin gene therapy.

Renal cell carcinoma (RCC) is characterized by high vascular endothelial growth factor (VEGF) production and, consequently, excessive angiogenesis. Several strategies have been developed to target angiogenesis as a method for treating metastatic RCC (mRCC). Endostatin (ES) is a C-terminal fragment of collagen XVIII that has antiangiogenic activity. The aim of this study was to investigate the predictive value of circulating VEGF-A in a murine model of mRCC after ES gene therapy. ES therapy did not affect the levels of collagen XVIII/ES or ES in the tissue. The circulating level of ES was increased in the control and ES-treated groups (normal vs. control, P<0.05 and ES-treated vs. control, P<0.001), and the intratumoral vessels were significantly decreased (ES-treated vs. control, P<0.05). ES therapy decreased the VEGF mRNA levels. The tissue and circulating levels of VEGF in the control group were significantly higher than normal (P<0.01 and P<0.05, respectively). Treatment with ES significantly reduced the VEGF concentrations in both compartments (P<0.001 for tissue and P<0.05 for plasma). Our findings indicate that in addition to the directly targeted tumor vessels, ES exerts its antitumor effect by down-regulating VEGF gene expression in renal tumor cells. Additionally, our findings point to the predictive value of VEGF for ES therapy.

Collagen XVIII/endostatin expression in experimental endotoxemic acute renal failure

Acute renal failure (ARF) is a frequent complication of Gram-negative sepsis, with a high risk of mortality. Lipopolysaccharide (LPS)-induced ARF is associated with hemodynamic changes that are strongly influenced by the overproduction of nitric oxide (NO) through the cytokine-mediated up-regulation of inducible NO synthase. LPS-induced reductions in systemic vascular resistance paradoxically culminate in renal vasoconstriction. Collagen XVIII is an important component of the extracellular matrix expressed in basement membranes. Its degradation by matrix metalloproteases, cathepsins and elastases results in the formation of endostatin, claimed to have antiangiogenic activity and to be a prominent vasorelaxing agent. We evaluated the expression of endostatin/collagen XVIII in an endotoxemic ARF model. ARF was induced in C57BL/6 mice by intraperitoneal injection of LPS (10 mg/kg) followed by sacrifice 4 and 12 h later. Kidney tissue was the source of RNA and protein and the subject of histological analysis. As early as 4 h after LPS administration, blood urea, creatinine and NO levels were significantly increased compared to control. Endostatin/collagen XVIII mRNA levels were 0.71 times lower than sham-inoculated mice 4 h after LPS inoculation, returning to normal levels 12 h after LPS inoculation. Immunohistological examination revealed that acute injury caused by LPS leads to an increase of endostatin basement membrane staining in association with the decrease of CD31 endothelial basement membrane staining. These results indicate that in the early phase of endotoxemic ARF the endostatin levels were not regulated by gene expression, but by the metabolism of collagen XVIII.

Collagen XVIII/endostatin expression in experimental endotoxemic acute renal failure.

Acute renal failure (ARF) is a frequent complication of Gram-negative sepsis, with a high risk of mortality. Lipopolysaccharide (LPS)-induced ARF is associated with hemodynamic changes that are strongly influenced by the overproduction of nitric oxide (NO) through the cytokine-mediated up-regulation of inducible NO synthase. LPS-induced reductions in systemic vascular resistance paradoxically culminate in renal vasoconstriction. Collagen XVIII is an important component of the extracellular matrix expressed in basement membranes. Its degradation by matrix metalloproteases, cathepsins and elastases results in the formation of endostatin, claimed to have antiangiogenic activity and to be a prominent vasorelaxing agent. We evaluated the expression of endostatin/collagen XVIII in an endotoxemic ARF model. ARF was induced in C57BL/6 mice by intraperitoneal injection of LPS (10 mg/kg) followed by sacrifice 4 and 12 h later. Kidney tissue was the source of RNA and protein and the subject of histological analysis. As early as 4 h after LPS administration, blood urea, creatinine and NO levels were significantly increased compared to control. Endostatin/collagen XVIII mRNA levels were 0.71 times lower than sham-inoculated mice 4 h after LPS inoculation, returning to normal levels 12 h after LPS inoculation. Immunohistological examination revealed that acute injury caused by LPS leads to an increase of endostatin basement membrane staining in association with the decrease of CD31 endothelial basement membrane staining. These results indicate that in the early phase of endotoxemic ARF the endostatin levels were not regulated by gene expression, but by the metabolism of collagen XVIII.

Novel pathogenic mutations and skin biopsy analysis in Knobloch syndrome.

PURPOSE: To facilitate future diagnosis of Knobloch syndrome (KS) and better understand its etiology, we sought to identify not yet described COL18A1 mutations in KS patients. In addition, we tested whether mutations in this gene lead to absence of the COL18A1 gene product and attempted to better characterize the functional effect of a previously reported missense mutation. METHODS: Direct sequencing of COL18A1 exons was performed in KS patients from four unrelated pedigrees. We used immunofluorescent histochemistry in skin biopsies to evaluate the presence of type XVIII collagen in four KS patients carrying two already described mutations: c.3277C>T, a nonsense mutation, and c.3601G>A, a missense mutation. Furthermore, we determined the binding properties of the mutated endostatin domain p.A1381T (c.3601G>A) to extracellular matrix proteins using ELISA and surface plasmon resonance assays. RESULTS: We identified four novel mutations in COL18A1, including a large deletion involving exon 41. Skin biopsies from KS patients revealed lack of type XVIII collagen in epithelial basement membranes and blood vessels. We also found a reduced affinity of p.A1381T endostatin to some extracellular matrix components. CONCLUSIONS: COL18A1 mutations involved in Knobloch syndrome have a distribution bias toward the coding exons of the C-terminal end. Large deletions must also be considered when point mutations are not identified in patients with characteristic KS phenotype. We report, for the first time, lack of type XVIII collagen in KS patients by immunofluorescent histochemistry in skin biopsy samples. As a final point, we suggest the employment of this technique as a preliminary and complementary test for diagnosis of KS in cases when mutation screening either does not detect mutations or reveals mutations of uncertain effect, such as the p.A1381T change.

COL18A1 is highly expressed during human adipocyte differentiation and the SNP c.1136C > T in its "frizzled" motif is associated with obesity in diabetes type 2 patients.

Collagen XVIII can generate two fragments, NC11-728 containing a frizzled motif which possibly acts in Wnt signaling and Endostatin, which is cleaved from the NC1 and is a potent inhibitor of angiogenesis. Collagen XVIII and Wnt signaling have recently been associated with adipogenic differentiation and obesity in some animal models, but not in humans. In the present report, we have shown that COL18A1 expression increases during human adipogenic differentiation. We also tested if polymorphisms in the Frizzled (c.1136C>T; Thr379Met) and Endostatin (c.4349G>A; Asp1437Asn) regions contribute towards susceptibility to obesity in patients with type 2 diabetes (113 obese, BMI > or =30; 232 non-obese, BMI < 30) of European ancestry. No evidence of association was observed between the allele c.4349G>A and obesity, but we observed a significantly higher frequency of homozygotes c.1136TT in obese (19.5%) than in non-obese individuals (10.9%) [P = 0.02; OR = 2.0 (95%CI: 1.07-3.73)], suggesting that the allele c.1136T is associated to obesity in a recessive model. This genotype, after controlling for cholesterol, LDL cholesterol, and triglycerides, was independently associated with obesity (P = 0.048), and increases the chance of obesity in 2.8 times. Therefore, our data suggest the involvement of collagen XVIII in human adipogenesis and susceptibility to obesity.

COL18A1 is highly expressed during human adipocyte differentiation and the SNP c.1136C > T in its "frizzled" motif is associated with obesity in diabetes type 2 patients

Collagen XVIII can generate two fragments, NC11-728 containing a frizzled motif which possibly acts in Wnt signaling and Endostatin, which is cleaved from the NC1 and is a potent inhibitor of angiogenesis. Collagen XVIII and Wnt signaling have recently been associated with adipogenic differentiation and obesity in some animal models, but not in humans. In the present report, we have shown that COL18A1 expression increases during human adipogenic differentiation. We also tested if polymorphisms in the Frizzled (c.1136C>T; Thr379Met) and Endostatin (c.4349G>A; Asp1437Asn) regions contribute towards susceptibility to obesity in patients with type 2 diabetes (113 obese, BMI =30; 232 non-obese, BMI < 30) of European ancestry. No evidence of association was observed between the allele c.4349G>A and obesity, but we observed a significantly higher frequency of homozygotes c.1136TT in obese (19.5 percent) than in non-obese individuals (10.9 percent) [P = 0.02; OR = 2.0 (95 percentCI: 1.07-3.73)], suggesting that the allele c.1136T is associated to obesity in a recessive model. This genotype, after controlling for cholesterol, LDL cholesterol, and triglycerides, was independently associated with obesity (P = 0.048), and increases the chance of obesity in 2.8 times. Therefore, our data suggest the involvement of collagen XVIII in human adipogenesis and susceptibility to obesity.

Colágeno XVIII pode gerar dois fragmentos, um correspondendo à região NC11-728 contendo o motivo ''frizzled'', o qual possivelmente atua na sinalização Wnt, e outro correspondendo a Endostatina, que é clivada a partir da região NC1 e é uma potente inibidora de angiogênese. Colágeno XVIII e a via de sinalização Wnt foram recentemente associados à diferenciação adipogênica e obesidade em alguns modelosanimais, porém ainda não em humanos. No presente trabalho, mostramos que os níveis de expressão gênica do COL18A1 aumentam durante o processo de diferenciação adipogênica em humanos. Também testamos se polimorfismos localizados no motivo ''Frizzled'' (c.1136C > T; Thr379Met) e na região da Endostatina (c.4349G > A; Asp1437Asn) contribuem na predisposição a obesidade em pacientes com diabetes tipo 2. (113 obesos, BMI > 30; 232 não-obesos, BMI < 30) de ancestralidade Européia. Nenhuma evidência de associação entre o alelo c.4349G > A e obesidade foi observada, contudo, observamos uma freqüência significativamente maior de homozigotos c.1136TT em obesos (19.5 por cento) do que em não-obesos (10.9 por cento)[P = 0.02; OR = 2.0 (95 por centoCI: 1.07-3.73)], sugerindo que o alelo c.1136T está associado com obesidade conforme ummodelo recessivo. Este genótipo manteve-se associado à obesidade (P = 0.048) mesmo após o controle das variáveis colesterol, LDL e triglicérides, e confere um risco 2.8 vezes maior de obesidade. Portanto, nossos dados sugerem o envolvimento do colágeno XVIII em adipogênese humana e predisposição a obesidade.

Angiotensin I-converting enzyme inhibitor peptides derived from the endostatin-containing NC1 fragment of human collagen XVIII.

Extracellular matrix and soluble plasma proteins generate peptides that regulate biological activities such as cell growth, differentiation and migration. Bradykinin, a peptide released from kininogen by kallikreins, stimulates vasodilatation and endothelial cell proliferation. Various classes of substances can potentiate these biological actions of bradykinin. Among them, the best studied are bradykinin potentiating peptides (BPPs) derived from snake venom, which can also strongly inhibit angiotensin I-converting enzyme (ACE) activity. We identified and synthesized sequences resembling BPPs in the vicinity of potential proteolytic cleavage sites in the collagen XVIII molecule, close to endostatin. These peptides were screened as inhibitors of human recombinant wild-type ACE containing two intact functional domains; two full-length ACE mutants containing only a functional C- or N-domain catalytic site; and human testicular ACE, a natural form of the enzyme that only contains the C-domain. The BPP-like peptides inhibited ACE in the micromolar range and interacted preferentially with the C-domain. The proteolytic activity involved in the release of BPP-like peptides was studied in human serum and human umbilical-vein endothelial cells. The presence of enzymes able to release these peptides in blood led us to speculate on a physiological mechanism for the control of ACE activities.

Mutations in collagen 18A1 and their relevance to the human phenotype.

Collagen XVIII, a proteoglycan, is a component of basement membranes (BMs). There are three distinct isoforms that differ only by their N-terminal, but with a specific pattern of tissue and developmental expression. Cleavage of its C-terminal produces endostatin, an inhibitor of angiogenesis. In its N-terminal, there is a frizzled motif which seems to be involved in Wnt signaling. Mutations in this gene cause Knobloch syndrome KS), an autosomal recessive disorder characterized by vitreoretinal and macular degeneration and occipital encephalocele. This review discusses the effect of both rare and polymorphic alleles in the human phenotype, showing that deficiency of one of the collagen XVIII isoforms is sufficient to cause KS and that null alleles causing deficiency of all collagen XVIII isoforms are associated with a more severe ocular defect. This review besides illustrating the functional importance of collagen XVIII in eye development and its structure maintenance throughout life, it also shows its role in other tissues and organs, such as nervous system and kidney.

Mutations in collagen 18A1 (COL18A1) and their relevance to the human phenotype

Colágeno XVIII, uma proteoglicana, é um componente das membranas basais (MBs). Existem três isoformas distintas que diferem apenas na região N-terminal, mas que apresentam um padrão específico de expressão nos diferentes tecidos e durante o desenvolvimento. A clivagem da região C-terminal produz endostatina, um inibidor de angiogênese. Na sua região N-terminal, há um motivo "frizzled'', o qual parece estar envolvido com a sinalização de Wnt. Mutações no gene COL18A1 causam a síndrome de Knobloch (SK), uma condição de herança autossômica recessiva caracterizada por degeneração vítreo - retiniana, degeneração de mácula e encefalocele occipital. Esta revisão discute o efeito tanto de alelos raros como polimórficos no fenótipo, mostrando que deficiência de uma das isoformas de colágeno XVIII é suficiente para causar SK e que alelos nulos causando deficiência de todas as isoformas de colágeno XVIII estão associadas a alterações oculares mais graves. Esta revisão, além de ilustrar a importância funcional do colágeno XVIII no desenvolvimento do olho e na manutenção de sua estrutura, também mostra que esta proteína tem um papel funcional importante em outros tecidos e órgão, como no sistema nervoso central e rim.

Characterization of human collagen XVIII promoter 2: interaction of Sp1, Sp3 and YY1 with the regulatory region and a SNP that increases transcription in hepatocytes.

Different levels of Collagen XVIII expression have been associated with several pathological processes such as cancer, liver fibrosis, diabetic retinopathy and Alzheimer's disease. Understanding the transcriptional regulation of Collagen XVIII might elucidate some pathways related to the progression of these diseases. The promoter 2 of COL18A1 gene is poorly understood and is responsible for the transcription of this gene in several adult tissues such as liver, eyes and brain. This study focused upon characterization of cis-regulatory elements interacting with human COL18A1 promoter 2 and identification of SNPs in this region in different ethnic groups. Our results show that there are five conserved regions (I to V) between human and mouse promoter 2 and that the human COL18A1 core promoter is located between nucleotides -186 and -21. Sp1 and Sp3 bind to conserved regions I and V, while Sp3 and YY1 interact with region II. We have verified that the SNP at position -700 (T>G) is embedded in two common haplotypes, which have different frequencies between European and African descendents. The allele -700G increases transcription and binding for a still unknown transcription factor. SNP -700 affects Sp3 and YY1 interaction with this region, even though it is not part of these transcription factors' predicted binding sites. Therefore, our results show for the first time that Sp3 and YY1 interact with human COL18A1 promoter 2, and that nucleotide -700 is part of a binding motif for a still unknown TF that is involved in the expression of this gene in hepatocytes. In addition, we also confirm the involvement of Sp1 in the regulation of this gene.

Evidence of neuronal migration disorders in Knobloch syndrome: clinical and molecular analysis of two novel families.

Knobloch syndrome is an autosomal recessive disease characterized by the early onset of severe myopia, vitreoretinal degeneration with retinal detachment, macular abnormalities, and midline encephalocele, mainly in the occipital region. Intra and interfamilial variability is present since the encephalocele is not found in all patients, and the degree of myopia is variable. Analysis of the associated malformations suggests alterations during early neuroectodermal morphogenesis. Only 24 cases have been reported. Recently, the gene responsible for the syndrome, mapped to 21q22.3, was identified. The present study reports on four new cases, revealing the existence of neuronal migratory defects associated with the disorder for the first time.