Genetic analysis of typical wet-type age-related macular degeneration and polypoidal choroidal vasculopathy in Japanese population

Age-related macular degeneration (AMD) is a common cause of blindness in the elderly. Caucasian patients are predominantly affected by the dry form of AMD, whereas Japanese patients have predominantly the wet form of AMD and/or polypoidal choroidal vasculopathy (PCV). Although genetic association in the 10q26 (ARMS2/HTRA1) region has been established in many ethnic groups for dry-type AMD, typical wet-type AMD, and PCV, the contribution of the 1q32 (CFH) region seem to differ among these groups. Here we show a single nucleotide polymorphism (SNP) in the ARMS2/HTRA1 locus is associated in the whole genome for Japanese typical wet-type AMD (rs10490924: , OR = 4.16) and PCV (rs10490924: , OR = 2.72) followed by CFH (rs800292: , OR = 2.08; , OR = 2.00), which differs from previous studies in Caucasian populations. Moreover, a SNP (rs2241394) in complement component C3 gene showed significant association with PCV (, OR = 3.47). We conclude that dry-type AMD, typical wet-type AMD, and PCV have both common and distinct genetic risks that become apparent when comparing Japanese versus Caucasian populations.

Electronic supplementary material

The online version of this article (doi:10.1007/s12177-009-9047-1) contains supplementary material, which is available to authorized users.     

Prevalence and Genetic Characteristics of Geographic Atrophy among Elderly Japanese with Age-Related Macular Degeneration

Objective

To investigate the prevalence and genetic characteristics of geographic atrophy (GA) among elderly Japanese with advanced age-related macular degeneration (AMD) in a clinic-based study.

Methods

Two-hundred and ninety consecutive patients with advanced AMD were classified into typical neovascular AMD, polypoidal choroidal vasculopathy (PCV), retinal angiomatous proliferation (RAP) or geographic atrophy (GA). Genetic variants of ARMS2 A69S (rs10490924) and CFH I62V (rs800292) were genotyped using TaqMan Genotyping Assays. The clinical and genetic characteristics were compared between patients with and without GA.

Results

The number of patients diagnosed as having typical neovascular AMD, PCV, RAP and GA were 98 (33.8%), 151 (52.1%), 22 (7.5%) and 19 (6.6%), respectively. Of 19 patients with GA, 13 patients (68.4%) had unilateral GA with exudative AMD in the contralateral eye. Patients with GA were significantly older, with a higher prevalence of reticular pseudodrusen, bilateral involvement of advanced AMD and T-allele frequency of ARMS2 A69S compared with those with typical AMD and PCV; although there were no differences in the genetic and clinical characteristics among patients with GA and RAP.

Conclusions

The prevalence of GA was 6.6% among elderly Japanese with AMD. Patients with GA and RAP exhibited genetic and clinical similarities.     

Effect of High-Density Lipoprotein Metabolic Pathway Gene Variations and Risk Factors on Neovascular Age-Related Macular Degeneration and Polypoidal Choroidal Vasculopathy in China

Purpose

To investigate the effect of genetic variants in the high-density lipoprotein (HDL) metabolic pathway and risk factors on neovascular age-related macular degeneration (nAMD) and polypoidal choroidal vasculopathy (PCV) in China.

Methods

A total of 742 Chinese subjects, including 221 controls, 230 cases with nAMD, and 291 cases with PCV, were included in the present study. Five single nucleotide polymorphisms (SNPs) from three genes in the HDL metabolic pathway (HDLMP) including cholesteryl ester transfer protein (CETP), hepatic lipase (LIPC) and lipoprotein lipase (LPL) were genotyped in all study subjects with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Risk factors including gender, hypertension, hyperlipidemia, diabetes mellitus, and coronary artery disease were identified. Chi-square tests or Fisher’s exact tests were applied to discover associations between SNPs and risk factors for PCV and nAMD. Gene-gene interactions and gene-environment interactions were evaluated by the multifactor-dimensionality reduction (MDR) method.

Results

CETP rs3764261 were significantly associated with an increased risk for PCV (odds ratio (OR) = 1.444, P = 0.0247). LIPC rs1532085 conferred an increased risk for PCV (OR = 1.393, P = 0.0094). We found no association between PCV and LPL rs12678919, LIPC rs10468017 or CETP rs173539. No association was found between five SNPs with nAMD. Regarding risk factors, females were found to have significantly decreased risks for both PCV and nAMD (P = 0.006 and 0.001, respectively). Coronary artery disease (CAD) was a risk factor in PCV patients but played a protective role in nAMD patients. Hyperlipidemia was associated with PCV but not with nAMD. Neither hypertension nor diabetes mellitus was associated with PCV or nAMD. The MDR analysis revealed that a three-locus model with rs12678919, rs1532085, and gender was the best model for nAMD, while a five-locus model consisting of rs10468017, rs3764261, rs1532085, gender, and hyperlipidemia was best for PCV.

Conclusion

Our large-sample study suggested that CETP rs3764261 conferred an increased risk for PCV. We also first found the association between rs1532085 and PCV. The result of present study also showed that gender and CAD are associated with PCV and nAMD. Significant association was found between hyperlipidemia and PCV but not nAMD.     

Cumulative association between age-related macular degeneration and less studied genetic variants in PLEKHA1/ARMS2/HTRA1: a meta and gene-cluster analysis

The objective of this study is to examine the cumulative effect of the less studied genetic variants in PLEKHA1/ARMS2/HTRA1 on age-related macular degeneration (AMD). We performed an extensive literature search for studies on the association between AMD and the less studied genetic variants in PLEKHA1/ARMS2/HTRA1. Multiple meta-analyses were performed to evaluate the association between individual genetic variants and AMD. A gene-cluster analysis was used to investigate the cumulative effect of these less studied genetic variants on AMD. A total of 23 studies from 20 published papers met the eligibility criteria and were included in our analyses. Several genetic variants in the gene cluster are significantly associated with AMD in our meta-analyses or in individual studies. Gene-cluster analysis reveals a strong cumulative association between these genetic variants in this gene cluster and AMD (p < 10^?5). However, two previously suspected SNPs in ARMS2, including rs2736911, the SNP having the largest number of studies in our meta-analyses; and rs3793917, the SNP with the largest sample size, were not significantly associated with AMD (both p’s > 0.12). Sensitivity analyses reveal significant association of AMD with rs2736911 in Chinese but not in Caucasian, with c.372_815del443ins54 in Caucasian but not in Chinese, and with rs1049331 in both ethnic groups. These less studied genetic variants have a significant cumulative effect on wet AMD. Our study provides evidence of the joint contribution of genetic variants in PLEKHA1/ARMS2/HTRA1 to AMD risk, in addition to the two widely studied genetic variants whose association with AMD was well established.     

rs5888 Variant of SCARB1 Gene Is a Possible Susceptibility Factor for Age-Related Macular Degeneration

Major genetic factors for age-related macular degeneration (AMD) have recently been identified as susceptibility risk factors, including variants in the CFH gene and the ARMS2 LOC387715/HTRA1locus. Our purpose was to perform a case-control study in two populations among individuals who did not carry risk variants for CFHY402H and LOC387715 A69S (ARMS2), called “study” individuals, in order to identify new genetic risk factors. Based on a candidate gene approach, we analyzed SNP rs5888 of the SCARB1 gene, coding for SRBI, which is involved in the lipid and lutein pathways. This study was conducted in a French series of 1241 AMD patients and 297 controls, and in a North American series of 1257 patients with advanced AMD and 1732 controls. Among these individuals, we identified 61 French patients, 77 French controls, 85 North American patients and 338 North American controls who did not carry the CFH nor ARMS2 polymorphisms. An association between AMD and the SCARB1 gene was seen among the study subjects. The genotypic distribution of the rs5888 polymorphism was significantly different between cases and controls in the French population (p<0.006). Heterozygosity at the rs5888 SNP increased risk of AMD compared to the CC genotypes in the French study population (odds ratio (OR) = 3.5, CI95%: 1.4–8.9, p<0.01) and after pooling the 2 populations (OR = 2.9, 95% CI: 1.6–5.3, p<0.002). Subgroup analysis in exudative forms of AMD revealed a pooled OR of 3.6 for individuals heterozygous for rs5888 (95% CI: 1.7–7.6, p<0.0015). These results suggest the possible contribution of SCARB1, a new genetic factor in AMD, and implicate a role for cholesterol and antioxidant micronutrient (lutein and vitamin E) metabolism in AMD.     

Overexpression of HTRA1 Leads to Down-Regulation of Fibronectin and Functional Changes in RF/6A Cells and HUVECs

Multiple genetic studies have suggested that high-temperature requirement serine protease (HTRA1) is associated with polypoidal choroidal vasculopathy (PCV). To date, no functional studies have investigated the biological effect of HTRA1 on vascular endothelial cells, essential vascular components involved in polypoidal vascular abnormalities and arteriosclerosis-like changes. In vitro studies were performed to investigate the effect of HTRA1 on the regulation of fibronectin, laminin, vascular endothelial growth factor (VEGF), platelet derived growth factor receptor (PDGFR) and matrix metalloparoteinases 2 (MMP-2) and the role of HTRA1 in choroid-retina endothelial (RF/6A) and human umbilical vein endothelial (HUVEC) cells. Lentivirus-mediated overexpression of HTRA1 was used to explore effects of the protease on RF/6A and HUVEC cells in vitro. HTRA1 overexpression inhibited the proliferation, cell cycle, migration and tube formation of RF/6A and HUVEC cells, effects that might contribute to the early stage of PCV pathological lesions. Fibronectin mRNA and protein levels were significantly down-regulated following the upregulation of HTRA1, whereas the expressions of laminin, VEGF and MMP-2 were unaffected by alterations in HTRA1 expression. The decreased biological function of vascular endothelial cells and the degradation of extracellular matrix proteins, such as fibronectin, may be involved in a contributory role for HTRA1 in PCV pathogenesis.     

High temperature requirement factor A1 (HTRA1) gene regulates angiogenesis through transforming growth factor-β family member growth differentiation factor 6

Genome-wide association study (GWAS) has identified genetic variants in the promoter region of the high temperature requirement factor A1 (HTRA1) gene associated with age-related macular degeneration (AMD). As a secreted serine protease, HTRA1 has been reported to interact with members of the transforming growth factor-β (TGF-β) family and regulate their signaling pathways. Growth differentiation factor 6 (GDF6), a member of the TGF-β family, is involved in ectoderm patterning and eye development. Mutations in GDF6 have been associated with abnormal eye development that may result in microphthalmia and anophthalmia. In this report, we identified a single nucleotide polymorphism (SNP) rs6982567 A/G near the GDF6 gene that is significantly associated with AMD (p value = 3.54 × 10(-8)). We demonstrated that the GDF6 AMD risk allele (rs6982567 A) is associated with decreased expression of the GDF6 and increased expression of HTRA1. Similarly, the HTRA1 AMD risk allele (rs10490924 T) is associated with decreased GDF6 and increased HTRA1 expression. We observed decreased vascular development in the retina and significant up-regulation of GDF6 gene in the RPE layer, retinal and brain tissues in HTRA1 knock-out (htra1(-/-)) mice as compared with the wild-type counterparts. Furthermore, we showed enhanced SMAD signaling in htra1(-/-) mice. Our data suggests a critical role of HTRA1 in the regulation of angiogenesis via TGF-β signaling and identified GDF6 as a novel disease gene for AMD.     

CFH, VEGF, and PEDF genotypes and the response to intravitreous injection of bevacizumab for the treatment of age-related macular degeneration

We determined whether there is an association between complement factor H (CFH), high-temperature requirement A-1 (HTRA1), vascular endothelial growth factor (VEGF), and pigment epithelium-derived factor (PEDF) genotypes and the response to treatment with a single intravitreous injection of bevacizumab for age-related macular degeneration (AMD). Eighty-three patients with exudative AMD treated by bevacizumab injection were genotyped for three single nucleotide polymorphisms (SNPs; rs800292, rs1061170, rs1410996) in the CFH gene, a rs11200638-SNP in the HTRA1 gene, three SNPs (rs699947, rs1570360, rs2010963) in the VEGF gene, and four SNPs (rs12150053, rs12948385, rs9913583, rs1136287) in the PEDF gene using a TaqMan assay. The CT genotype (heterozygous) of CFH-rs1061170 was more frequently represented in nonresponders in vision than TT genotypes (nonrisk allele homozygous) at the time points of 1 and 3 months, while there was no CC genotype (risk allele homozygous) in our study cohort (p = 7.66 × 10⁻³, 7.83 × 10⁻³, respectively). VEGF-rs699947 was also associated with vision changes at 1 month and PEDF-rs1136287 at 3 months (p = 5.11 × 10⁻³, 2.05 × 10⁻², respectively). These variants may be utilized for genetic biomarkers to estimate visual outcomes in the response to intravitreal bevacizumab treatment for AMD.     

Association of Htra1 gene polymorphisms with the risk of developing AMD in Iranian population

Background:

Half of the cases of vision loss in people under 60 years of age have been attributed to age-related macular degeneration (AMD). This is a multifactorial disease with late onset. It has been demonstrated that many different genetic loci are implicated in the risk of developing AMD in different populations. In the current study, we investigated the association of high-temperature ‎requirement A-1 (HTRA1) gene polymorphisms with the risk of developing AMD in the Iranian population.

Methods:

Genomic DNA samples were extracted from 120 patients with AMD and 120 healthy age- and sex-matched controls. A 385 base-pair fragment of the HTRA1 gene promoter region was amplified using the polymerase chain reaction (PCR) technique and sequenced. The frequencies of the alleles were calculated and statistical analysis was performed using SPSS software.

Results:

Our study demonstrated that the rate of polymorphisms rs11200638 -625 G>A and rs2672598 -487T>C were significantly greater in AMD patients than in healthy controls from the Iranian population.

Conclusions:

The results of our study indicate that HTRA1 gene promoter region polymorphisms are associated with the risk of developing AMD in the Iranian population.Key Words: HTRA1, Single Nucleotide Polymorphisms, Macular Degeneration, Iran     

Interleukin-1β Level Is Increased in Vitreous of Patients with Neovascular Age-Related Macular Degeneration (nAMD) and Polypoidal Choroidal Vasculopathy (PCV)

PurposeTo examine the expression of pro-interleukin-1β (pro-IL-1β) and interleukin-1β (IL-1β) in the vitreous body of patients with neovascular age-related macular degeneration(nAMD), polypoidal choroidal vasculopathy (PCV), proliferative diabetic retinopathy (PDR), retinal vein occlusion (RVO) or Eales’ disease to further elucidate the role of IL-1β and inflammation in the pathogenesis of neovascular retinal disease.DesignProspective clinical laboratory investigation study.MethodsAll patients enrolled had vitreous hemorrhage due to nAMD, PCV, PDR, RVO or Eales’ disease that required vitrectomy. Patients were excluded for any history of active intraocular inflammation, or other ophthalmic surgery besides vitrectomy. Control samples were obtained from patients with idiopathic macular epiretinal membrane. A total of fifty vitreous samples were collected from patient during vitrectomy. Pro-IL-1β and IL-1β expression were measured by enzyme-linked immunosorbent assay (ELISA). Results were analyzed statistically using nonparametric tests.ResultsExpression of pro-IL-1β protein was increased by 2.83-fold and 9.19-fold in PCV and nAMD vitreous samples relative to control, respectively. Expression of IL-β protein was increased by 10-fold and 4.83-fold in PCV and nAMD vitreous samples relative to control, respectively.ConclusionsOur results demonstrate that expression of pro-IL-1β and IL-1β proteins is higher in PCV and nAMD. The roles of pro-IL-1β and IL-1β as inflammatory mediators in the development of PCV and nAMD may be associated with photoreceptor degeneration and neovascularization which necessitates further study.     

Glycogen synthase kinase 3β represses MYOGENIN function in alveolar rhabdomyosarcoma

MYOGENIN is a member of the muscle regulatory factor family that orchestrates an obligatory step in myogenesis, the terminal differentiation of skeletal muscle cells. A paradoxical feature of alveolar rhabdomyosarcoma (ARMS), a prevalent soft tissue sarcoma in children arising from cells with a myogenic phenotype, is the inability of these cells to undergo terminal differentiation despite the expression of MYOGENIN. The chimeric PAX3-FOXO1 fusion protein which results from a chromosomal translocation in ARMS has been implicated in blocking cell cycle arrest, preventing myogenesis from occurring. We report here that PAX3-FOXO1 enhances glycogen synthase kinase 3β (GSK3β) activity which in turn represses MYOGENIN activity. MYOGENIN is a GSK3β substrate in vitro on the basis of in vitro kinase assays and MYOGENIN is phosphorylated in ARMS-derived RH30 cells. Constitutively active GSK3β(S9A) increased the level of a phosphorylated form of MYOGENIN on the basis of western blot analysis and this effect was reversed by neutralization of the single consensus GSK3β phosphoacceptor site by mutation (S160/164A). Congruently, GSK3β inhibited the trans-activation of an E-box reporter gene by wild-type MYOGENIN, but not MYOGENIN with the S160/164A mutations. Functionally, GSK3β repressed muscle creatine kinase (MCK) promoter activity, an effect which was reversed by the S160/164A mutated MYOGENIN. Importantly, GSK3β inhibition or exogenous expression of the S160/164A mutated MYOGENIN in ARMS reduced the anchorage independent growth of RH30 cells in colony-formation assays. Thus, sustained GSK3β activity represses a critical regulatory step in the myogenic cascade, contributing to the undifferentiated, proliferative phenotype in alveolar rhabdomyosarcoma (ARMS).     

Copy number variation of age-related macular degeneration relevant genes in the Korean population

PurposeStudies that analyzed single nucleotide polymorphisms (SNP) in various genes have shown that genetic factors are strongly associated with age-related macular degeneration (AMD) susceptibility. Copy number variation (CNV) may be an additional type of genetic variation that contributes to AMD pathogenesis. This study investigated CNV in 4 AMD-relevant genes in Korean AMD patients and control subjects.MethodsFour CNV candidate regions located in AMD-relevant genes (VEGFA, ARMS2/HTRA1, CFH and VLDLR), were selected based on the outcomes of our previous study which elucidated common CNVs in the Asian populations. Real-time PCR based TaqMan Copy Number Assays were performed on CNV candidates in 273 AMD patients and 257 control subjects.ResultsThe predicted copy number (PCN, 0, 1, 2 or 3+) of each region was called using the CopyCaller program. All candidate genes except ARMS2/HTRA1 showed CNV in at least one individual, in which losses of VEGFA and VLDLR represent novel findings in the Asian population. When the frequencies of PCN were compared, only the gain in VLDLR showed significant differences between AMD patients and control subjects (p = 0.025). Comparisons of the raw copy values (RCV) revealed that 3 of 4 candidate genes showed significant differences (2.03 vs. 1.92 for VEGFA, p<0.01; 2.01 vs. 1.97 for CFH, p<0.01; 1.97 vs. 2.01, p<0.01 for ARMS2/HTRA1).ConclusionCNVs located in AMD-relevant genes may be associated with AMD susceptibility. Further investigations encompassing larger patient cohorts are needed to elucidate the role of CNV in AMD pathogenesis.     

The Association of Elastin Gene Variants with Two Angiographic Subtypes of Polypoidal Choroidal Vasculopathy

Objective

To compare the association of elastin (ELN) gene variants between two different angiographic phenotypes of polypoidal choroidal vasculopathy (PCV).

Methods

We included 411 treatment-naïve PCV patients and 350 controls in the present study. PCV was classified into two phenotypes (152 Type 1 and 259 Type 2) according to the presence or absence of feeding vessels found in indocyanine-green angiography. Single nucleotide polymorphisms (SNPs) in the ELN region including rs868005, rs884843, rs2301995, rs13239907 and rs2856728 were genotyped using TaqMan Genotyping Assays.

Results

In the allelic association analyses, rs868005 showed the strongest association with Type 2 PCV (allelic odds ratio 1.56; p = 7.4x10^-6), while no SNP was significantly associated with Type 1 PCV. Genotype association analyses revealed the significant association of rs868005 with Type 2 PCV in log additive model and predominant model (odds ratio 1.75; p = 1.5x10^-6 and odds ratio 1.60; p = 0.0044, respectively), but not with Type 1 PCV. These findings were further corroborated by another control group in the literature.

Conclusions

There may be significantly different associations in genetic variants of elastin between two angiographic phenotypes of PCV.     

Detection and Clinical Significance of Intratumoral EGFR Mutational Heterogeneity in Chinese Patients with Advanced Non-Small Cell Lung Cancer

Purpose

This study evaluated occurrence and potential clinical significance of intratumoral EGFR mutational heterogeneity in Chinese patients with non-small cell lung cancer (NSCLC).

Materials and Methods

Eighty-five stage IIIa-IV NSCLC patients who had undergone palliative surgical resection were included in this study. Of these, 45 patients carried EGFR mutations (group-M) and 40 patients were wild-type (group-W). Each tumor sample was microdissected to yield 28–34 tumor foci and Intratumoral EGFR mutation were determined using Denaturing High Performance Liquid Chromatography (DHPLC) and Amplification Refractory Mutation System (ARMS). EGFR copy numbers were measured using fluorescence in situ hybridization (FISH).

Results

Microdissection yielded 1,431 tumor foci from EGFR mutant patients (group-M) and 1,238 foci from wild-type patients (group-W). The EGFR mutant frequencies in group-M were 80.6% (1,154/1,431) and 87.1% (1,247/1,431) using DHPLC and ARMS, respectively. A combination of EGFR-mutated and wild-type cells was detected in 32.9% (28/85) of samples by DHPLC and 28.2% (24/85) by ARMS, supporting the occurrence of intratumoral heterogeneity. Thirty-one patients (36.5%) were identified as EGFR FISH-positive. Patients harboring intratumoral mutational heterogeneity possessed lower EGFR copy numbers than those tumors contained mutant cells alone (16.7% vs. 71.0%, P<0.05). Among 26 patients who had received EGFR-TKIs, the mean EGFR mutation content was higher in patients showing partial response (86.1%) or stable disease (48.7%) compared with patients experiencing progressive disease (6.0%) (P = 0.001). There also showed relationship between progression-free survival (PFS) and different content of EGFR mutation groups (pure wild type EGFR, EGFR mutation with heterogeneity and pure mutated EGFR) (P = 0.001).

Conclusion

Approximately 30% of patients presented intratumoral EGFR mutational heterogeneity, accompanying with relatively low EGFR copy number. EGFR mutant content was correlated with the response and prognosis of EGFR-TKIs.     

Differential DNA methylation identified in the blood and retina of AMD patients

Age-related macular degeneration (AMD) is a major cause of blindness in the western world. While genetic studies have linked both common and rare variants in genes involved in regulation of the complement system to increased risk of development of AMD, environmental factors, such as smoking and nutrition, can also significantly affect the risk of developing the disease and the rate of disease progression. Since epigenetics has been implicated in mediating, in part, the disease risk associated with some environmental factors, we investigated a possible epigenetic contribution to AMD. We performed genome-wide DNA methylation profiling of blood from AMD patients and controls. No differential methylation site reached genome-wide significance; however, when epigenetic changes in and around known GWAS-defined AMD risk loci were explored, we found small but significant DNA methylation differences in the blood of neovascular AMD patients near age-related maculopathy susceptibility 2 (ARMS2), a top-ranked GWAS locus preferentially associated with neovascular AMD. The methylation level of one of the CpG sites significantly correlated with the genotype of the risk SNP rs10490924, suggesting a possible epigenetic mechanism of risk. Integrating genome-wide DNA methylation analysis of retina samples with and without AMD together with blood samples, we further identified a consistent, replicable change in DNA methylation in the promoter region of protease serine 50 (PRSS50). These methylation changes may identify sites in novel genes that are susceptible to non-genetic factors known to contribute to AMD development and progression.     

Genomic aspects of age-related macular degeneration

Age-related macular degeneration (AMD) is a major late-onset posterior eye disease that causes central vision to deteriorate among elderly populations. The predominant lesion of AMD is the macula, at the interface between the outer retina and the inner choroid. Recent advances in genetics have revealed that inflammatory and angiogenic pathways play critical roles in the pathophysiology of AMD. Genome-wide association studies have identified ARMS2/HTRA1 and CFH as major AMD susceptibility genes. Genetic studies for AMD will contribute to the prevention of central vision loss, the development of new treatment, and the maintenance of quality of vision for productive aging.     

Transposition of IS 117, the 2.5 kb Streptomyces coelicolor A3(2) 'minicircle': roles of open reading frames and origin of tandem insertions

IS 117 is a 2527 bp transposable element from Streptomyces coelicolor A3(2) with a circular transposition intermediate. Disruption of 0RF1 of IS 117, presumed to encode a transposase, abolished transposition. Deletion or mutation of 0RF2 and 0RF3, which overlap each other on opposite strands of IS 117, caused a c. 20-fold reduction in integration frequency of the circular form of IS 117 into the Streptomyces lividans chromosome or into the preferred chromosomal target site cloned on a plasmid in transformation experiments. In contrast, inactivation of ORF2/3 did not significantly influence transposition of IS 117 derivatives from an already integrated state in the chromosome to the preferred target site cloned on a plasmid. 0RF2 mutants apparently excised readily from the S. lividans chromosome, whereas excision of integrated wild-type IS 117 derivatives to yield the unoccupied site was not detected; presumably, therefore, the circular transposition intermediate normally arises replicatively. Attempts to promote integration of a plasmid carrying the attachment site of IS 117 by providing the ORF1 product in trans were unsuccessful. Most transformation of S. lividans with circular IS 117 derivatives yielded tandem chromosomal insertions, which arose by co-transformation rather than dimerization of a monomeric insert. Typically, two to three transforming elements gave a transformed strain, suggesting a local concentration of transposase as a limit on integration.     

Detrimental role for human high temperature requirement serine protease A1 (HTRA1) in the pathogenesis of intervertebral disc (IVD) degeneration

Human HTRA1 is a highly conserved secreted serine protease that degrades numerous extracellular matrix proteins. We have previously identified HTRA1 as being up-regulated in osteoarthritic patients and as having the potential to regulate matrix metalloproteinase (MMP) expression in synovial fibroblasts through the generation of fibronectin fragments. In the present report, we have extended these studies and investigated the role of HTRA1 in the pathogenesis of intervertebral disc (IVD) degeneration. HTRA1 mRNA expression was significantly elevated in degenerated disc tissue and was associated with increased protein levels. However, these increases did not correlate with the appearance of rs11200638 single nucleotide polymorphism in the promoter region of the HTRA1 gene, as has previously been suggested. Recombinant HTRA1 induced MMP production in IVD cell cultures through a mechanism critically dependent on MEK but independent of IL-1β signaling. The use of a catalytically inactive mutant confirmed these effects to be primarily due to HTRA1 serine protease activity. HTRA1-induced fibronectin proteolysis resulted in the generation of various sized fragments, which when added to IVD cells in culture, caused a significant increase in MMP expression. Furthermore, one of these fragments was identified as being the amino-terminal fibrin- and heparin-binding domain and was also found to be increased within HTRA1-treated IVD cell cultures as well as in disc tissue from patients with IVD degeneration. Our results therefore support a scenario in which HTRA1 promotes IVD degeneration through the proteolytic cleavage of fibronectin and subsequent activation of resident disc cells.