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Cerebral amyloid angiopathy (CAA) is an age-related condition and a major cause of intracerebral hemorrhage and cognitive decline that shows close links with Alzheimer's disease (AD). CAA is characterized by the aggregation of amyloid-β (Aβ) peptides and formation of Aβ deposits in the brain vasculature resulting in a disruption of the angioarchitecture. Capillaries are a critical site of Aβ pathology in CAA type 1 and become dysfunctional during disease progression. Here, applying an advanced protocol for the isolation of parenchymal microvessels from post-mortem brain tissue combined with liquid chromatography tandem mass spectrometry (LC–MS/MS), we determined the proteomes of CAA type 1 cases (n = 12) including a patient with hereditary cerebral hemorrhage with amyloidosis-Dutch type (HCHWA-D), and of AD cases without microvascular amyloid pathology (n = 13) in comparison to neurologically healthy controls (n = 12). ELISA measurements revealed microvascular Aβ1-40 levels to be exclusively enriched in CAA samples (mean: > 3000-fold compared to controls). The proteomic profile of CAA type 1 was characterized by massive enrichment of multiple predominantly secreted proteins and showed significant overlap with the recently reported brain microvascular proteome of patients with cerebral autosomal-dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL), a hereditary cerebral small vessel disease (SVD) characterized by the aggregation of the Notch3 extracellular domain. We found this overlap to be largely attributable to the accumulation of high-temperature requirement protein A1 (HTRA1), a serine protease with an established role in the brain vasculature, and several of its substrates. Notably, this signature was not present in AD cases. We further show that HTRA1 co-localizes with Aβ deposits in brain capillaries from CAA type 1 patients indicating a pathologic recruitment process. Together, these findings suggest a central role of HTRA1-dependent protein homeostasis in the CAA microvasculature and a molecular connection between multiple types of brain microvascular disease. Supplementary Information The online version contains supplementary material available at 10.1186/s40478-021-01303-6.

Background Single-variant associations with age-related macular degeneration (AMD), one of the most prevalent causes of irreversible vision loss worldwide, have been studied extensively. However, because of a lack of refinement of these associations, there remains considerable ambiguity regarding what constitutes genetic risk and/or protection for this disease, and how genetic combinations affect this risk. In this study, we consider the two most common and strongly AMD-associated loci, the CFH-CFHR5 region on chromosome 1q32 (Chr1 locus) and ARMS2/HTRA1 gene on chromosome 10q26 (Chr10 locus). Results By refining associations within the CFH-CFHR5 locus, we show that all genetic protection against the development of AMD in this region is described by the combination of the amino acid-altering variant CFH I62V (rs800292) and genetic deletion of CFHR3/1. Haplotypes based on CFH I62V, a CFHR3/1 deletion tagging SNP and the risk variant CFH Y402H are associated with either risk, protection or neutrality for AMD and capture more than 99% of control- and case-associated chromosomes. We find that genetic combinations of CFH-CFHR5 haplotypes (diplotypes) strongly influence AMD susceptibility and that individuals with risk/protective diplotypes are substantially protected against the development of disease. Finally, we demonstrate that AMD risk in the ARMS2/HTRA1 locus is also mitigated by combinations of CFH-CFHR5 haplotypes, with Chr10 risk variants essentially neutralized by protective CFH-CFHR5 haplotypes. Conclusions Our study highlights the importance of considering protective CFH-CFHR5 haplotypes when assessing genetic susceptibility for AMD. It establishes a framework that describes the full spectrum of AMD susceptibility using an optimal set of single-nucleotide polymorphisms with known functional consequences. It also indicates that protective or preventive complement-directed therapies targeting AMD driven by CFH-CFHR5 risk haplotypes may also be effective when AMD is driven by ARMS2/HTRA1 risk variants.

Congenital cataract is the leading cause of blindness among children worldwide. Patients with posterior subcapsular congenital cataract (PSC) in the central visual axis can result in worsening vision and stimulus deprivation amblyopia. However, the pathogenesis of PSC remains unclear. This study aims to explore the functional regulation and mechanism of HTRA1 in human lens epithelial cells (HLECs). HTRA1 was significantly downregulated in the lens capsules of children with PSC compared to normal controls. HTRA1 is a suppression factor of transforming growth factor-β (TGF-β) signalling pathway, which plays a key role in cataract formation. The results showed that the TGF-β/Smad signalling pathway was activated in the lens tissue of PSC. The effect of HTRA1 on cell proliferation, migration and apoptosis was measured in HLECs. In primary HLECs, the downregulation of HTRA1 can promote the proliferation and migration of HLECs by activating the TGF-β/Smad signalling pathway and can significantly upregulate the TGF-β/Smad downstream target genes FN1 and α-SMA. HTRA1 was also knocked out in the eyes of C57BL/6J mice via adeno-associated virus-mediated RNA interference. The results showed that HTRA1 knockout can significantly upregulate p-Smad2/3 and activate the TGF-β/Smad signalling pathway, resulting in abnormal proliferation and irregular arrangement of lens epithelial cells and leading to the occurrence of subcapsular cataract. To conclude, HTRA1 was significantly downregulated in children with PSC, and the downregulation of HTRA1 enhanced the proliferation and migration of HLECs by activating the TGF-β/Smad signalling pathway, which led to the occurrence of PSC.

BACKGROUND Age-related macular degeneration (AMD) mainly affects the central region of retina and has many late-stage manifestations. OBJECTIVES Age-related macular degeneration is a leading cause of irreversible blindness in older people. The main feature of AMD is retinal pigment epithelium (RPE) degeneration. In this study, we aimed to explore the influence of HTRA1 expression on the proliferation and migration of RPE cells. MATERIAL AND METHODS Human ARPE-19 cells were transfected with an HTRA1 overexpression lentivirus or HTRA1 siRNA to silence HtrA1 expression. Quantitave reverse-transcription polymerase chain reaction (qRT-PCR) and western blotting were used to verify the relative level of HTRA1 mRNA and expression of HTRA1 protein of transfected human ARPE-19 cells. The MTT clone formation and transwell assays were used to confirm the effect of HTRA1 expression on the proliferation, colony forming ability and migration of ARPE-19 cells. RESULTS The proliferation capacity (shown as optical density value) of ARPE-19 cells in the HTRA1-overexpressing group at culture times of 24 h and 48 h were 0.595 ±0.032 and 0.867 ±0.037 respectively, which were much higher than in the mock group. However, the proliferative capacity of cells in the HTRA1-silenced group decreased with increasing time of culture, compared with the mock group. The number of cloned and migrating cells in the HTRA1-overexpressing group were much higher than in the mock group, whereas the numbers in the HTRA1-silenced group were significantly lower. CONCLUSIONS Overexpression of HTRA1 promotes proliferation and migration of RPE cells, which can help maintain the function of sensory neurons in the retina. Therefore, HTRA1 may be a suitable target for AMD treatments.

Introduction Vestibular schwannoma (VS) is a benign intracranial tumor in which the underlying genetics is largely uncertain, apart from mutations in the tumor suppressor gene NF2. Alternative tumorigenic mechanisms have been proposed, including a recurrent in-frame fusion transcript of the HTRA1 and SH3PXD2A genes. The gene product of the SH3PXD2A-HTRA1 fusion has been shown to promote proliferation, invasion and resistance to cell death in vitro and tumor growth in vivo. The aim of this study was to replicate the findings and to investigate the frequency of this fusion gene in another cohort of vestibular schwannoma patients. Methods The SH3PXD2A-HTRA1 transcript was synthesized in vitro using PCR and used as a positive control to assess the sensitivity of a real-time PCR assay. This real-time PCR assay was used to search for the presence of the fusion transcript in 121 Norwegian sporadic VS patients. Results The real-time PCR assay showed a high sensitivity and was able to detect as low as ~ 5 copies of the fusion transcript. Out of the 121 investigated tumors, only 1 harbored the SH3PXD2A-HTRA1 fusion. Conclusion Even though the SH3PXD2A-HTRA1 fusion has been shown to be a driver of tumorigenesis, our results suggest that it is a rare event in our VS patients. Further investigation is warranted in order to elucidate whether our results represent an extreme, and if the fusion is present also in other neoplasms.

Purpose: Age-related maculopathy susceptibility 2 (ARMS2) is considered the most enigmatic of the genes for age-related macular degeneration (AMD). We investigated the phenotypic course and spectrum of AMD for the risk haplotype at the ARMS2 and high-temperature requirement A serine peptidase 1 (HTRA1) locus in a large European consortium. Design: Pooled analysis of 4 case-control and 6 cohort studies. Participants: Individuals (N = 17 204) aged 55 years or older participating in the European Eye Epidemiology consortium. Methods: Age-related macular degeneration features and macular thickness were determined on multimodal images; data on genetics and phenotype were harmonized. Risks of AMD features for rs3750486 genotypes at the ARMS2/HTRA1 locus were determined by logistic regression and were compared with a genetic risk score (GRS) of 19 variants at the complement pathway. Lifetime risks were estimated with Kaplan–Meier analyses in population-based cohorts. Main Outcome Measures: Age-related macular degeneration features and stage. Results: Of 2068 individuals with late AMD, 64.7% carried the ARMS2/HTRA1 risk allele. For homozygous carriers, the odds ratio (OR) of geographic atrophy was 8.6 (95% confidence interval [CI], 6.5–11.4), of choroidal neovascularization (CNV) was 11.2 (95% CI, 9.4–13.3), and of mixed late AMD was 12.2 (95% CI, 7.3–20.6). Cumulative lifetime risk of late AMD ranged from 4.4% for carriers of the nonrisk genotype to 9.4% and 26.8% for heterozygous and homozygous carriers. The latter received the diagnosis of late AMD 9.6 years (95% CI, 8.0–11.2) earlier than carriers of the nonrisk genotype. The risk haplotype was not associated with hard or soft drusen < 125 μm (OR, 1.2; 95% CI, 0.9–1.7), but risks increased significantly for soft drusen ≥ 125 μm (OR, 2.1; 95% CI, 1.5–3.0), up to an OR of 7.2 (95% CI, 3.8–13.8) for reticular pseudodrusen. Compared with persons with a high GRS for complement, homozygous carriers of ARMS2/HTRA1 showed a higher risk of CNV (OR, 4.1; 95% CI, 3.2–5.4); risks of other characteristics were not different. Conclusions: Carriers of the risk haplotype at ARMS2/HTRA1 have a particularly high risk of late AMD at a relatively early age. Data suggest that risk variants at ARMS2/HTRA1 act as a strong catalyst of progression once early signs are present. The phenotypic spectrum resembles that of complement genes, only with higher risks of CNV.

Purpose: To identify the association between single-nucleotide polymorphisms (SNPs) in CFH, ARMS2, HTRA1, CFB, C2, and C3 genes and exudative age-related macular degeneration (AMD) in a Spanish population. Methods: In 187 exudative AMD patients and 196 healthy controls (61% women, mean age 75 years), 12 SNPs as risk factors for AMD in CFH (rs1410996, rs1061170, r380390), ARMS2 (rs10490924, rs10490923), HTRA1 (rs11200638), CFB (rs641153), C2 (rs547154, rs9332739), and C3 (rs147859257, rs2230199, rs1047286) genes were analyzed. Results: The G allele was the most frequent in CFH gene (rs1410996) with a 7-fold increased risk of AMD (OR 7.69, 95% CI 3.17–18.69), whereas carriers of C allele in CFH (rs1061170) showed a 3-fold increased risk for AMD (OR 3.22, 95% CI 1.93–5.40). In CFH (rs380390), the presence of G allele increased the risk for AMD by 2-fold (OR 2.52, 95% CI 1.47–4.30). In ARMS2 (rs10490924), the T-allele was associated with an almost 5-fold increased risk (OR 5.49, 95% CI 3.23–9.31). The A allele in HTRA1 (rs11200638) was more prevalent in AMD versus controls (OR 6.44, 95% CI 3.62–11.47). In C2 gene (rs9332739) the presence of C increased risk for AMD by 3-fold (OR 3.10, 95% CI 1.06–9.06). Conclusion: SNPs in CFH, ARMS2, HTRA1, and C2 genes were associated in our study with an increased risk for exudative AMD in Spanish patients.

Background: Copy number variations (CNVs) play an important role in the genetic etiology of various neurological disorders, including Alzheimer’s disease (AD). Type 2 diabetes mellitus (T2DM) and major depressive disorder (MDD) were shown to have share mechanisms and signaling pathways with AD. Objective: We aimed to assess CNVs regions that may harbor genes contributing to AD, T2DM, and MDD in 67 Saudi familial and sporadic AD patients, with no alterations in the known genes of AD and genotyped previously for APOE. Methods: DNA was analyzed using the CytoScan-HD array. Two layers of filtering criteria were applied. All the identified CNVs were checked in the Database of Genomic Variants (DGV). Results: A total of 1086 CNVs (565 gains and 521 losses) were identified in our study. We found 73 CNVs harboring genes that may be associated with AD, T2DM or MDD. Nineteen CNVs were novel. Most importantly, 42 CNVs were unique in our studied cohort existing only in one patient. Two large gains on chromosomes 1 and 13 harbored genes implicated in the studied disorders. We identified CNVs in genes that encode proteins involved in the metabolism of amyloid-β peptide (AGRN, APBA2, CR1, CR2, IGF2R, KIAA0125, MBP, RER1, RTN4R, VDR and WISPI) or Tau proteins (CACNAIC, CELF2, DUSP22, HTRA1 and SLC2A14). Conclusion: The present work provided information on the presence of CNVs related to AD, T2DM, and MDD in Saudi Alzheimer’s patients.

Purpose AMD genetic studies have revealed various genetic loci as causal to AMD pathology. We have described the genetic complexity of Indian AMD by describing the interaction of genotypes and subsequent changes in protein expression under the influence of environmental factors. This can be utilized to enhance the diagnostic and therapeutic efficacy in AMD patients. Design Genotype association was studied in 464 participants (AMD =277 & controls = 187) for eight genetic variants and their corresponding protein expression Methods SNP analysis and protein expression analysis was carried out in AMD and controls in tandem with longitudinal assessment of protein levels during the course of AMD pathology. ANCOVA and contrast analysis were used to examine the genotypic interactions and corresponding alterations in protein levels. In order to identify the important genetic variants Logistic Regression (LR) modeling was carried out and to authenticate the model Area under the Receiver Operating Characteristic curve (AUROC) were also computed. Results We have found genetic variants of rs5749482 (TIMP-3), rs11200638 (HTRA1), rs769449 (APOE) and rs6795735 (ADAMTS9) to be associated with AMD, concomitant with significant alterations of studied proteins levels. Analysis also revealed that the genetic interaction between APOE-HTRA1 genotypes and changes in LIPC levels (>6 pg/ug) by one unit change in SNP, play a crucial role in AMD. LR model suggested that the seven factors (including both genetic and environmental) can be utilized to predict the AMD cases with 88% efficacy and 95.6% AUROC. Conclusion Results suggest that diagnostic and therapeutic strategy for Indian AMD must include estimation of genetic interaction and concomitant changes in expression levels of proteins under influence of environmental factors.

The high temperature requirement A (HTRA) family of serine proteases mediates protein quality control. These proteins process misfolded proteins in several diseases including Alzheimer's disease (AD) and Parkinson's disease (PD). While their structures and activation mechanisms have been studied, the precise details of the regulation of their activity under physiological conditions have not been completely elucidated, partly due to the lack of suitable chemical probes. In the present study, we developed novel activity-based probes (ABPs) targeting the HTRAs and demonstrated their utility in the monitoring and quantification of changes in enzyme activity in live cells. Using our probes, we found the activity of HTRA1 to be highly elevated in an AD-like cell-based model. We also observed the active HTRA2 in live cells by using a mitochondrion-targeted probe. We believe that our probes can serve as a useful tool to study the role of human HTRAs in neurodegenerative diseases.

Significance Genome-wide association studies have identified genetic variation at the ARMS2/HTRA1 locus as a risk factor for the development and progression of age-related macular degeneration (AMD). We have developed a potent anti-HtrA1 Fab inhibitor of HtrA1 proteolytic activity in the retina as a potential therapeutic for treating AMD. A set of proteomic analytical tools was established to characterize HtrA1 activity and discover in vivo HtrA1 substrates. These efforts led to the identification of an eye-specific and clinically applicable pharmacodynamic biomarker of anti-HtrA1 Fab activity. Analysis of HtrA1-mediated cleavage of Dickkopf-related protein 3 in the aqueous humor of patients with geographic atrophy provided evidence of anti-HtrA1 Fab activity and information on duration of activity in a phase 1 study., Genetic polymorphisms in the region of the trimeric serine hydrolase high-temperature requirement 1 (HTRA1) are associated with increased risk of age-related macular degeneration (AMD) and disease progression, but the precise biological function of HtrA1 in the eye and its contribution to disease etiologies remain undefined. In this study, we have developed an HtrA1-blocking Fab fragment to test the therapeutic hypothesis that HtrA1 protease activity is involved in the progression of AMD. Next, we generated an activity-based small-molecule probe (ABP) to track target engagement in vivo. In addition, we used N-terminomic proteomic profiling in preclinical models to elucidate the in vivo repertoire of HtrA1-specific substrates, and identified substrates that can serve as robust pharmacodynamic biomarkers of HtrA1 activity. One of these HtrA1 substrates, Dickkopf-related protein 3 (DKK3), was successfully used as a biomarker to demonstrate the inhibition of HtrA1 activity in patients with AMD who were treated with the HtrA1-blocking Fab fragment. This pharmacodynamic biomarker provides important information on HtrA1 activity and pharmacological inhibition within the ocular compartment.

Purpose Genetic studies have identified the association of some single-nucleotide polymorphisms (SNPs) with polypoidal choroidal vasculopathy (PCV), but little is known about whether these SNPs are related to PCV clinical features as well. We performed this study to examine the association of 12 SNPs with PCV clinical phenotypes. Methods Sixty-nine PCV eyes of 69 patients were included. Genomic DNA was extracted from peripheral blood. Agilent SureSelect Human ALL Exon V6 was used to sequence the 12 SNPs previously reported to associate with PCV. Baseline best-corrected visual acuity (BCVA), sub-foveal choroidal thickness (SFCT), choroid maximum vascular diameter (MVD), choroidal vascular hyperpermeability (CVH), and greatest linear dimension (GLD) of entire lesion were measured and compared between patients of different genotypes. Fisher’s exact test and Mann-Whitney U test were mainly used to compare categorical variables and continuous variables respectively. Results HTRA1 rs2293870 was a protective factor of PCV or AMD in the fellow eye (P = 0.040) and was related with greater SFCT in PCV eye after multiple linear regression (P = 0.043). C3 rs17030 was associated with smaller GLD (P = 0.033). CFH rs2274700 was related to lower MVD (P = 0.043) and was a protective factor for CVH (P = 0.034). Conclusion Multiple PCV-associated SNPs are associated with PCV clinical phenotypes. The involvement of several synonymous SNPs calls for further research on the role of transcriptional alterations and trans-regulation of distant signaling pathways in PCV pathogenesis.

Contains fulltext : 219118.pdf (Publisher’s version ) (Open Access) PURPOSE: Age-related macular degeneration (AMD) is a degenerative disease of the macula, often leading to progressive vision loss. The rate of disease progression can vary among individuals and has been associated with multiple risk factors. In this review, we provide an overview of the current literature investigating phenotypic, demographic, environmental, genetic, and molecular risk factors, and propose the most consistently identified risk factors for disease progression in AMD based on these studies. Finally, we describe the potential use of these risk factors for personalised healthcare. RECENT FINDINGS: While phenotypic risk factors such as drusen and pigment abnormalities become more important to predict disease progression during the course of the disease, demographic, environmental, genetic and molecular risk factors are more valuable at earlier disease stages. Demographic and environmental risk factors such as age and smoking are consistently reported to be related to disease progression, while other factors such as sex, body mass index (BMI) and education are less often associated. Of all known AMD variants, variants that are most consistently reported with disease progression are rs10922109 and rs570618 in CFH, rs116503776 in C2/CFB/SKIV2L, rs3750846 in ARMS2/HTRA1 and rs2230199 in C3. However, it seems likely that other AMD variants also contribute to disease progression but to a lesser extent. Rare variants have probably a large effect on disease progression in highly affected families. Furthermore, current prediction models do not include molecular risk factors, while these factors can be measured accurately in the blood. Possible promising molecular risk factors are High-Density Lipoprotein Cholesterol (HDL-C), Docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA), zeaxanthin and lutein. SUMMARY: Phenotypic, demographic, environmental, genetic and molecular risk factors can be combined in prediction models to predict disease progression, but the selection of the proper risk factors for personalised risk prediction will differ among individuals and is dependent on their current disease stage. Future prediction models should include a wider set of genetic variants to determine the genetic risk more accurately, and rare variants should be taken into account in highly affected families. In addition, adding molecular factors in prediction models may lead to preventive strategies and personalised advice.

High temperature requirement A1 (HTRA1) is a serine protease playing a modulatory role in various cell processes, particularly in the regulation of transforming growth factor-β (TGF-β) signaling. A deleterious role in late-onset cerebral small vessel diseases (CSVDs) of heterozygous HTRA1 mutations, otherwise causative in homozygosity of cerebral autosomal recessive arteriopathy with subcortical infarcts and leukoencephalopathy, was recently suggested. However, the pathomechanism of these heterozygous mutations is still undefined. Our aim is to evaluate the expression profile and activity of HTRA1 on TGF-β signaling in fibroblasts from four subjects carrying the HTRA1 heterozygous mutations-p.E42Dfs*173, p.A321T, p.G295R, and p.Q151K. We found a 50% reduction of HTRA1 expression in HTRA1 mutation carriers compared to the control. Moreover, we showed no changes in TGF-β signaling pathway downstream intermediate, Phospho Smad2/3. However, we found overexpression of genes involved in the extracellular matrix formation in two heterozygous HTRA1 carriers. Our results suggest that each heterozygous HTRA1 missense mutation displays a different and peculiar HTRA1 expression pattern and that CSVD phenotype may also result from 50% of HTRA1 expression.

Homozygous or compound heterozygous mutations in the high-temperature requirement A serine protease 1 gene (HTRA1) cause cerebral autosomal recessive arteriopathy with subcortical infarcts and leukoencephalopathy, a very rare hereditary cerebral small-vessel disease (SVD). Recently, the relationship between some heterozygous HTRA1 mutations, most of which are missense, and the occurrence of cerebral SVD has been reported. We herein report a patient with cerebral SVD carrying a heterozygous nonsense p.R302X mutation in HTRA1. This patient had a family history of cerebral infarction. This report suggests that a heterozygous p.R302X mutation in HTRA1 causes an autosomal dominant cerebral SVD.

To gain insight into the aetiology of posterior subcapsular congenital cataract from the perspective of transcriptional changes, we conducted an mRNA sequencing analysis of the lenses in posterior subcapsular congenital cataract patients and in normal children. There were 1533 differentially expressed genes from 19,072 genes in the lens epithelial cells of the posterior subcapsular congenital cataract patients compared to in the normal controls at a cut-off criteria of |log2 fold change| of >1 and a p-value of 1 and an FDR value of

Biallelic mutations in the high-temperature requirement A serine peptidase 1 (HTRA1) gene are known to cause an extremely rare cerebral autosomal recessive arteriopathy with subcortical infarcts and leukoencephalopathy (CARASIL), which belongs to the group of hereditary cerebral small vessel diseases and is mainly observed in the Japanese population. Even though this pathology is inherited in an autosomal recessive manner, recent studies have described symptomatic carriers with heterozygous HTRA1 mutations who have milder symptoms than patients with biallelic HTRA1 mutations. We present the case of a Lithuanian male patient who had a stroke at the age of 36, experienced several transient ischemic attacks, and developed an early onset, progressing dementia. These clinical symptoms were associated with extensive leukoencephalopathy, lacunar infarcts, and microbleeds based on brain magnetic resonance imaging (MRI). A novel heterozygous in-frame HTRA1 gene deletion (NM_002775.5:c.533_535del; NP_002766.1:p.(Lys178del)) was identified by next generation sequencing. The variant was consistent with the patient’s phenotype, which could not be explained by alternative causes, appeared highly deleterious after in silico analysis, and was not reported in the medical literature or population databases to date.

The mammalian high temperature requirement A (HtrA) proteins are a family of evolutionarily conserved serine proteases, consisting of four homologs (HtrA1-4) that are involved in many cellular processes such as growth, unfolded protein stress response and programmed cell death. In humans, while HtrA1, 2 and 3 are widely expressed in multiple tissues with variable levels, HtrA4 expression is largely restricted to the placenta with the protein released into maternal circulation during pregnancy. This limited expression sets HtrA4 apart from the rest of the family. All four HtrAs are active proteases, and their specific cellular and physiological roles depend on tissue type. The dysregulation of HtrAs has been implicated in many human diseases such as cancer, arthritis, neurogenerative ailments and reproductive disorders. This review first discusses HtrAs broadly and then focuses on the current knowledge of key molecular characteristics of individual human HtrAs, their similarities and differences and their reported physiological functions. HtrAs in other species are also briefly mentioned in the context of understanding the human HtrAs. It then reviews the distinctive involvement of each HtrA in various human diseases, especially cancer and pregnancy complications. It is noteworthy that HtrA4 expression has not yet been reported in any primary tumour samples, suggesting an unlikely involvement of this HtrA in cancer. Collectively, we accentuate that a better understanding of tissue-specific regulation and distinctive physiological and pathological roles of each HtrA will improve our knowledge of many processes that are critical for human health.

Background Biallelic pathogenic variants in HTRA1 cause CARASIL. More recently, monoallelic variants have been associated with the autosomal dominant disorder CADASIL2 but not all carriers develop disease manifestations. We describe the clinicoradiologic and mutation spectrum of four new CADASIL2 individuals. Methods Medical records at Mayo Clinic between 2013 and 2020 were retrospectively reviewed to identify patients with cerebral small vessel disease related to monoallelic HTRA1 variants. Results Four patients met the study inclusion criteria for cerebral small vessel disease related to HTRA1 monoallelic variants. The mean age at onset of first clinical stroke was 51.25 years (range 41–64 years). The mean disease duration was 6.5 years (range 4–12). All individuals had recurrent strokes within the duration of follow‐up with a mean number of strokes per patient being 5.5 (range 2–12). Three individuals had leukoencephalopathy with brain stem involvement. Microhemorrhages were seen on brain MRI in three patients. HTRA1 monoallelic variants identified in our cohort were missense variants in three patients and a novel frameshift variation in one patient. Interestingly, two of these missense variants were previously reported in an autosomal recessive pattern of inheritance and here are associated with a dominant effect. Conclusions Clinicoradiologic characteristics of heterozygous HTRA1‐related CSVD may overlap with sporadic CSVD. Heterozygous HTRA1 variants can contribute to dominant or recessive disease mechanisms., Clinicoradiologic features of heterozygous HTRA1‐related CSVD may overlap with sporadic CVSD. The presence of vascular risk factors and a noncontributory family history should not exclude late‐onset CSVD of inherited etiology. HTRA1 variants can be disease‐causing in both heterozygous and biallelic states, but so far, there are no defining variant characteristics to determine the pattern of inheritance.

A 44-year-old Caucasian man presented with seizures and cognitive impairment. He had marked retinal drusen, and MR brain scan showed features of cerebral small vessel disease; he was diagnosed with a leukoencephalopathy of uncertain cause. He died at the age of 46 years and postmortem brain examination showed widespread small vessel changes described as a vasculopathy of unknown cause. Seven years postmortem, whole-genome sequencing identified a homozygous nonsense HTRA1 mutation (p.Arg302Ter), giving a retrospective diagnosis of cerebral autosomal recessive arteriopathy with subcortical infarcts and leukoencephalopathy.

Degeneration of macular photoreceptors is a prominent characteristic of age-related macular degeneration (AMD) which leads to devastating and irreversible vision loss in the elderly population. In this exploratory study, the contribution of environmental factors on the progression of AMD pathology by probing the expression of candidate proteins was analyzed. Four hundred and sixty four participants were recruited in the study comprising of AMD (n = 277) and controls (n = 187). Genetics related data was analyzed to demonstrate the activities of daily living (ADL) by using regression analysis and statistical modeling, including contrast estimate, multinomial regression analysis in AMD progression. Regression analysis revealed contribution of smoking, alcohol, and sleeping hours on AMD by altered expression of IER-3, HTRA1, B3GALTL, LIPC and TIMP3 as compared to normal levels. Contrast estimate supports the gender polarization phenomenon in AMD by significant decreased expression of SLC16A8 and LIPC in control population which was found to be unaltered in AMD patients. The smoking, food habits and duration of night sleeping hours also contributed in AMD progression as evident from multinomial regression analysis. Predicted model (prediction estimate = 86.7%) also indicated the crucial role of night sleeping hours along with the decreased expression of TIMP-3, IER3 and SLC16A8. Results revealed an unambiguous role of environmental factors in AMD progression mediated by various regulatory proteins which might result in intermittent AMD phenotypes and possibly influence the outcome of anti-VEGF treatment.

The serine protease high-temperature requirement protein A1 (HtrA1) is associated with protein-misfolding disorders such as Alzheimer's disease and transforming growth factor b-induced protein (TGFBIp)-linked corneal dystrophy. In this study, using several biochemical and biophysical approaches, including recombinant protein expression, LC-MS/MS and 2D-PAGE analyses and thioflavin-T (ThT) fluorescence assays for amyloid fibril detection, and FTIR assays, we investigated the role of HtrA1 both in normal TGFBIp turnover and in corneal amyloid formation. We show that HtrA1 can cleave wild-type TGFBIp but prefers amyloidogenic variants. Corneal TGFBIp is extensively processed in healthy people, resulting in C-terminal degradation products spanning the FAS1-4 domain of TGFBIp. We show here that HtrA1 cleaves the wild-type FAS1-4 domain only inefficiently, whereas the amyloidogenic FAS1-4 mutations transform this domain into a considerably better HTRA1 substrate. Moreover, HtrA1 cleavage of the mutant FAS1-4 domains generated peptides capable of forming in vitro amyloid aggregates. Significantly, these peptides have been previously identified in amyloid deposits in vivo, supporting the idea that HtrA1 is a causative agent for TGFBIp-associated amyloidosis in corneal dystrophy. In summary, our results indicate that TGFBIp is an HtrA1 substrate and that some mutations in the gene encoding TGFBIp cause aberrant HtrA1-mediated processing that results in amyloidogenesis in corneal dystrophies.

Purpose The purpose of this study was to determine associations between macular drusen parameters derived from an automatic optical coherence tomography (OCT) algorithm, nonadvanced age-related macular degeneration (AMD) stage, and genetic variants. Methods Eyes classified as early or intermediate AMD with OCT imaging and genetic data were selected (n = 239 eyes). Drusen area and volume measurements were estimated using the Zeiss Cirrus advanced retinal pigment epithelium analysis algorithm in a perifoveal zone centered on the fovea. Associations between drusen measurements and common genetic variants in the complement and high-density lipoprotein (HDL) lipid pathways and the ARMS2/HTRA1 variant were calculated using generalized estimating equations and linear mixed models adjusting for age, sex, smoking, body mass index, and education. Results Drusen area ≥ the median was independently associated with a higher number of risk alleles for CFH risk score and risk variants in C3 and ARMS2/HTRA1 compared with eyes with no measurable drusen. Similar results were obtained for drusen volume. When all genes were analyzed in the same model, only CFH score and ARMS2/HTRA1 were associated with drusen measurements. HDL pathway genes were not significantly related to drusen parameters. Nonadvanced AMD stages were associated with OCT-derived drusen area and volume. Conclusions Variants in CFH and ARMS2/HTRA1, commonly associated with advanced AMD, were independently associated with an increase in drusen burden determined by OCT in an allele dose dependent manner, in eyes with early and intermediate AMD. Biomarkers such as a quantitative classification of nonadvanced AMD and other OCT-derived subphenotypes could provide earlier anatomic endpoints for clinical trials and facilitate the development of new therapies for AMD.

Objective To describe and compare two cases of North American and African patients who were diagnosed with HTRA1-related cerebral small vessel disease (CSVD) with homozygous and heterozygous mutations, respectively, in the linker domain of the HTRA1 gene. Materials and methods Case reports and literature review. Results A 49-year-old man from Mexico presented with recurrent lacunar strokes and memory loss. A 46-year-old woman from Eritrea presented with progressive memory loss. Neither patient had alopecia. MRI of the brain and spine in both patients showed leukoencephalopathy, microbleeds and spondylosis. Microbleeds along the subpial surfaces of the brainstem were only seen in the Mexican man. Genetic sequencing of HTRA1 gene revealed a novel homozygous mutation of p.A173S in the Mexican man supporting cerebral autosomal recessive arteriopathy with subcortical infarcts and leukoencephalopathy (CARASIL). A heterozygous mutation of p.V175M was detected in the African woman, which has not been reported in patients of African ethnicity. In reviewing literature, CARASIL patients with mutation in the linker domain are older at neurological symptom onset and more frequently presented with stroke compared to patients with non-linker domain mutations. In patients of HTRA1-CSVD from heterozygous mutations, male is more common. Conclusions HTRA1-related CSVD may be seen in patients of non-Asian ethnicity without alopecia. These case reports extend the clinical and radiographic spectrum of HTRA1-related CSVD.

Small linear motifs targeting protein interacting domains called PSD‐95/Dlg/ZO‐1 (PDZ) have been identified at the C terminus of the severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) proteins E, 3a, and N. Using a high‐throughput approach of affinity‐profiling against the full human PDZome, we identified sixteen human PDZ binders of SARS‐CoV‐2 proteins E, 3A, and N showing significant interactions with dissociation constants values ranging from 3 to 82 μm. Six of them (TJP1, PTPN13, HTRA1, PARD3, MLLT4, LNX2) are also recognized by SARS‐CoV while three (NHERF1, MAST2, RADIL) are specific to SARS‐CoV‐2 E protein. Most of these SARS‐CoV‐2 protein partners are involved in cellular junctions/polarity and could be also linked to evasion mechanisms of the immune responses during viral infection. Among the binders of the SARS‐CoV‐2 proteins E, 3a, or N, seven significantly affect viral replication under knock down gene expression in infected cells. This PDZ profiling identifying human proteins potentially targeted by SARS‐CoV‐2 can help to understand the multifactorial severity of COVID19 and to conceive effective anti‐coronaviral agents for therapeutic purposes., The E, 3a, and N proteins of the severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) carry a PSD‐95/Dlg/ZO‐1 (PDZ)‐binding motif allowing interaction with PDZ‐containing proteins in infected cells. A high‐throughput approach of affinity‐profiling against the full human PDZome identified 16 human PDZ binders of SARS‐CoV‐2 proteins, out of which seven significantly affect viral replication under knock down gene expression in infected cells. Most of them are involved in cellular junctions/polarity.

Cerebral small vessel disease (CSVD) is the most important cause of vascular cognitive impairment (VCI). Most CSVD cases are sporadic but familial monogenic forms of the disorder have also been described. Despite the variants identified, many CSVD cases remain unexplained genetically. We used whole-exome sequencing in an attempt to identify novel gene variants underlying CSVD. A cohort of 35 Finnish patients with suspected CSVD was analyzed. Patients were screened negative for the most common variants affecting function in NOTCH3 in Finland (p.Arg133Cys and p.Arg182Cys). Whole-exome sequencing was performed to search for a genetic cause of CSVD. Our study resulted in the detection of possibly pathogenic variants or variants of unknown significance in genes known to associate with CSVD in six patients, accounting for 17% of cases. Those genes included NOTCH3, HTRA1, COL4A1, and COL4A2. We also identified variants with predicted pathogenic effect in genes associated with other neurological or stroke-related conditions in seven patients, accounting for 20% of cases. This study supports pathogenic roles of variants in COL4A1, COL4A2, and HTRA1 in CSVD and VCI. Our results also suggest that vascular pathogenic mechanisms are linked to neurodegenerative conditions and provide novel insights into the molecular basis of VCI.

Cerebral amyloid angiopathy (CAA) is characterized by accumulation of amyloid β (Aβ) in walls of leptomeningeal vessels and cortical capillaries in the brain. The loss of integrity of these vessels caused by cerebrovascular Aβ deposits results in fragile vessels and lobar intracerebral hemorrhages. CAA also manifests with progressive cognitive impairment or transient focal neurological symptoms. Although development of therapeutics for CAA is urgently needed, the pathogenesis of CAA remains to be fully elucidated. In this review, we summarize the epidemiology, pathology, clinical and radiological features, and perspectives for future research directions in CAA therapeutics. Recent advances in mass spectrometric methodology combined with vascular isolation techniques have aided understanding of the cerebrovascular proteome. In this paper, we describe several potential key CAA-associated molecules that have been identified by proteomic analyses (apolipoprotein E, clusterin, SRPX1 (sushi repeat-containing protein X-linked 1), TIMP3 (tissue inhibitor of metalloproteinases 3), and HTRA1 (HtrA serine peptidase 1)), and their pivotal roles in Aβ cytotoxicity, Aβ fibril formation, and vessel wall remodeling. Understanding the interactions between cerebrovascular Aβ deposits and molecules that accumulate with Aβ may lead to discovery of effective CAA therapeutics and to the identification of biomarkers for early diagnosis.

Patients with primary mitral regurgitation (MR) may remain asymptomatic for many years. For unknown reasons, some shift from a compensated to a decompensated state and progress to fatal heart failure. To elucidate the genetic determinants of this process, we recruited 28 patients who underwent mitral valve surgery and stratified them into control, compensated MR, and decompensated MR groups. Tissue biopsies were obtained from the patients’ left ventricular (LV) lateral wall for a transcriptome-wide profiling of 64,769 probes to identify differentially expressed genes (DEGs). Using cutoff values at the 1% FDR significance level and sex- and age-adjusted regression models, we identified 12 significant DEGs (CTGF, MAP1B, SERPINE1, MYH9, MICAL2, MYO1D, CRY1, AQP7P3, HTRA1, PRSS23, IGFBP2, and FN1). The most significant gene was CTGF (adjusted R2 = 0.74, p = 1.80 × 10−8). We found that the majority of genes expressed in the more advanced decompensated MR group were pro-fibrotic genes associated with cardiac fibrosis. In particular, six pro-fibrotic genes (CTGF, SERPINE1, MYH9, HTRA1, PRSS23, and FN1) were overexpressed and enriched in pathways involved in ECM (extracellular matrix) protein remodeling. Therapeutic interventions that antagonize these six genes may slow the progression toward decompensated MR.

APOE ε4 is the major genetic risk factor for Alzheimer’s disease (AD). A precise role for apolipoprotein E (apoE) in the pathogenesis of the disease remains unclear in part due to its expression in multiple cell types of the brain. APOE is highly expressed in astrocytes and microglia, however its expression can also be induced in neurons under various conditions. The neuron-like cell line SK-N-SH is a useful model in the study of the cellular and molecular effects of apoE as it can be differentiated with retinoic acid to express and secrete high levels of apoE and it also shows the same apoE fragmentation patterns observed in the human brain. We previously found that apoE is cleaved into a 25-kDa fragment by high temperature-requirement serine protease A1 (HtrA1) in SK-N-SH cells. To further understand the endogenous functions of apoE, we used CRISPR/Cas9 to generate SK-N-SH cell lines with APOE expression knocked-down (KD). APOE KD cells showed lower APOE and HTRA1 expression than parental SK-N-SH cells but no overt differences in neuritogenesis or cell proliferation compared with the CRISPR/Cas9 control cells. This research shows that the loss of apoE and HtrA1 has a negligible effect on neuritogenesis and cell survival in SK-N-SH neuron-like cells.

White matter hyperintensities (WMH) are among the most common radiological abnormalities in the ageing population and an established risk factor for stroke and dementia. While common variant association studies have revealed multiple genetic loci with an influence on their volume, the contribution of rare variants to the WMH burden in the general population remains largely unexplored. We conducted a comprehensive analysis of this burden in the UK Biobank using publicly available whole-exome sequencing data (n up to 17 830) and found a splice-site variant in GBE1, encoding 1,4-alpha-glucan branching enzyme 1, to be associated with lower white matter burden on an exome-wide level [c.691+2T>C, β = −0.74, standard error (SE) = 0.13, P = 9.7 × 10−9]. Applying whole-exome gene-based burden tests, we found damaging missense and loss-of-function variants in HTRA1 (frequency of 1 in 275 in the UK Biobank population) to associate with an increased WMH volume (P = 5.5 × 10−6, false discovery rate = 0.04). HTRA1 encodes a secreted serine protease implicated in familial forms of small vessel disease. Domain-specific burden tests revealed that the association with WMH volume was restricted to rare variants in the protease domain (amino acids 204–364; β = 0.79, SE = 0.14, P = 9.4 × 10−8). The frequency of such variants in the UK Biobank population was 1 in 450. The WMH volume was brought forward by ∼11 years in carriers of a rare protease domain variant. A comparison with the effect size of established risk factors for WMH burden revealed that the presence of a rare variant in the HTRA1 protease domain corresponded to a larger effect than meeting the criteria for hypertension (β = 0.26, SE = 0.02, P = 2.9 × 10−59) or being in the upper 99.8% percentile of the distribution of a polygenic risk score based on common genetic variants (β = 0.44, SE = 0.14, P = 0.002). In biochemical experiments, most (6/9) of the identified protease domain variants resulted in markedly reduced protease activity. We further found EGFL8, which showed suggestive evidence for association with WMH volume (P = 1.5 × 10−4, false discovery rate = 0.22) in gene burden tests, to be a direct substrate of HTRA1 and to be preferentially expressed in cerebral arterioles and arteries. In a phenome-wide association study mapping ICD-10 diagnoses to 741 standardized Phecodes, rare variants in the HTRA1 protease domain were associated with multiple neurological and non-neurological conditions including migraine with aura (odds ratio = 12.24, 95%CI: 2.54–35.25; P = 8.3 × 10−5]. Collectively, these findings highlight an important role of rare genetic variation and the HTRA1 protease in determining WMH burden in the general population.

Cerebral autosomal recessive arteriopathy with subcortical infarcts and leukoencephalopathy (CARASIL) is caused by biallelic HTRA1 pathogenic variants. Recent studies have shown that heterozygous HTRA1 mutations are associated with autosomal dominant cerebral small vessel disease (CSVD). However, large studies evaluating heterozygous HTRA1 carriers are lacking and the genotype-phenotype correlation is unknown. This study aimed to describe these mutations to clarify factors playing a role in the clinical phenotype amongst these patients. We reported two unrelated families and performed a systematic review of all published cases of heterozygous HTRA1-related CSVD. The clinical phenotype severity was independently related to the pathogenicity score (CADD score; p 0.05) and mutation in the loop 3/loop D domains (p = 0.05); the pathogenicity score was also associated with exon distribution. More importantly, patients with mutations in exon 4 (p = 0.0001) or vascular risk factors (p 0.05) presented with more severe clinical symptoms. Thus, clinical phenotype severity is influenced by the mutation domain and vascular risk factors. Applying the pathogenicity score to predict clinical outcomes and adopting preventive measures against cerebral vascular risk factors is advantageous.

Calcium-selective transient receptor potential Vanilloid 6 (TRPV6) channels are expressed in fetal labyrinth trophoblasts as part of the feto&ndash, maternal barrier, necessary for sufficient calcium supply, embryo growth, and bone development during pregnancy. Recently, we have shown a less- compact labyrinth morphology of Trpv6-deficient placentae, and reduced Ca2+ uptake of primary trophoblasts upon functional deletion of TRPV6. Trpv6-/- trophoblasts show a distinct calcium-dependent phenotype. Deep proteomic profiling of wt and Trpv6-/- primary trophoblasts using label-free quantitative mass spectrometry leads to the identification of 2778 proteins. Among those, a group of proteases, including high-temperature requirement A serine peptidase 1 (HTRA1) and different granzymes are more abundantly expressed in Trpv6-/- trophoblast lysates, whereas the extracellular matrix protein fibronectin and the fibronectin-domain-containing protein 3A (FND3A) were markedly reduced. Trpv6-/-placenta lysates contain a higher intrinsic proteolytic activity increasing fibronectin degradation. Our results show that the extracellular matrix formation of the placental labyrinth depends on TRPV6, its deletion in trophoblasts correlates with the increased expression of proteases controlling the extracellular matrix in the labyrinth during pregnancy.

Background. Homozygous or compound heterozygous mutations in high temperature requirement serine peptidase A1 (HTRA1) gene are responsible for cerebral autosomal recessive arteriopathy with subcortical infarcts and leukoencephalopathy (CARASIL). Recently, increasing evidence has shown that heterozygous HTRA1 mutations are also associated with cerebral small vessel disease (CSVD) with an autosomal dominant pattern of inheritance. This study was aimed to analyze the genetic and clinical characteristics of HTRA1-related autosomal dominant CSVD. Methods. We presented three new Chinese cases of familial CSVD with heterozygous HTRA1 mutations and reviewed all clinical case reports and articles on HTRA1-related autosomal dominant CSVD included in PUBMED by the end of March 1, 2020. CARASIL probands with genetic diagnosis reported to date were also reviewed. The genetic and clinical characteristics of HTRA1-related autosomal dominant CSVD were summarized and analyzed by comparing with CARASIL. Results. Forty-four HTRA1-related autosomal dominant CSVD probands and 22 CARASIL probands were included. Compared with typical CARASIL, HTRA1-related autosomal dominant probands has a higher proportion of vascular risk factors (P

Background Cisplatin is a first-line drug for the treatment of human non-small cell lung cancer (NSCLC); however, the majority of patients will develop drug resistance after treatment. In order to overcome cisplatin resistance, it is important to understand the mechanisms underlying the resistance. Methods A gene microarray was used to screen for genes related to cisplatin resistance in NSCLC cell lines. Subsequently, the correlation between the HDAC, RXR and HtrA1 genes, in NSCLC, were verified using gene manipulation. Immunohistochemical staining was used to detect HDAC, RXR and HtrA1 expression in NSCLC specimens. Proliferation, migration and invasion assays were performed in vitro and in vivo to determine the role of the HDAC/RXR/HtrA1 signaling axis in cisplatin resistance, and luciferase reporter analysis and ChIP assays were performed to ascertain the mechanisms by which HDAC and RXR regulate the expression of HtrA1. Furthermore, in vitro and in vivo experiments were conducted in NSCLC cisplatin-resistant NSCLC to elucidate the effect of the low molecular weight compound, DW22, which targets the NSCLC cisplatin resistance HDAC/RXR/HtrA1 signaling pathway. Results HtrA1 was identified as a cisplatin resistance-related gene in NSCLC cells. The regulation of HtrA1 by HDAC and RXR significantly decreased the efficacy of cisplatin in NSCLC cells resistant to cisplatin. Immunohistochemistry results showed a negative relationship between HDAC1 and HtrA1, and a positive relationship between RXRα and HtrA1 in NSCLC patients’ tissues. Notably, the expression of HDAC1 and HtrA1 can be considered as biomarkers for the efficacy of platinum-based drugs and prognosis in NSCLC patients. Mechanistically, the heterodimers of the nuclear receptor RXR, in combination with the enzyme, HDAC, regulate the transcription of HtrA1 in NSCLC cells. The rescue of HtrA1 expression by dual targeting of HDAC and RXR with the compound, DW22, significantly inhibited the proliferation, migration and invasion of NSCLC cells resistant to cisplatin, and induced NSCLC cell apoptosis. Conclusion Our results indicate that HtrA1, a cisplatin resistance-related gene, is synergistically regulated by HDAC and RXR in NSCLC. Targeting the HDAC/RXR/HtrA1 signaling axis can rescue HtrA1 expression and reverse cisplatin resistance in NSCLC.

Introduction Altered placental expression of high temperature requirement factor A1 (HtrA1) is implicated in abnormal trophoblastic invasion and endothelial dysfunction in pre-eclampsia (PE). Serum levels of HtrA1 have been proposed as a novel biomarker to improve the prediction of PE. This study assesses serum HtrA1 levels in prospectively collected samples of women who developed PE compared to normotensive pregnancies. Methods This was a case-control study of serum HtrA1 levels in second and third trimester samples in women who later developed preterm or term PE compared to controls. Overall, 300 serum samples were drawn from a prospective observational study of adverse pregnancy outcomes in three different gestational age windows (19–24, 30-34 and 35–37 weeks) at the Fetal Medicine Research Institute, King's College Hospital, London. Serum HtrA1 levels were determined by enzyme-linked immunosorbent assay (ELISA) by a blinded laboratory professional. Median HtrA1 MoM values, adjusted for gestational age and maternal characteristics, were compared between cases and controls at each gestational age group. Results Women who later developed PE, compared to controls, had significantly higher maternal weight and more frequently had chronic hypertension or a history of PE in a previous pregnancy. In normotensive pregnancies, serum HtrA1 increased with increasing gestational age, whereas, in PE pregnancies HtrA1 levels remained stable, but were not significantly different from control pregnancies at any gestational age. Discussion Serum HtrA1 levels are not significantly different in women who develop PE compared to controls.

A minor haplotype of chromosome 10q26 accounts for much of the genetic risk of age-related macular degeneration (AMD). In this issue of Immunity, Beguier et al. demonstrate that carriers of the 10q26 AMD-risk haplotype overexpress the peptidase HTRA1, which in turns results in mononuclear phagocyte persistence in an immune privileged site and pathogenic inflammation.

The two most common genetic contributors to age-related macular degeneration (AMD), a leading cause of irreversible vision loss worldwide, are variants associated with CFH-CFHR5 on chromosome 1 (Chr1) and ARMS2/HTRA1 on chromosome 10 (Chr10). We sought to determine if risk and protective variants associated with these two loci drive differences in macular retinal thickness prior and subsequent to the onset of clinically observable signs of AMD. We considered 299 individuals (547 eyes) homozygous for risk variants or haplotypes on Chr1 or Chr10 exclusively (Chr1-risk and Chr10-risk, respectively) or homozygous for a neutral haplotype (Chr1-neu), for the protective I62 tagged haplotype (Chr1-prot-I62) or for the protection conferring CFHR3/1 deletion haplotype (Chr1-prot-del) on Chr1 without any risk alleles on Chr10. Among eyes with no clinically observable signs of AMD, the deletion of CFHR3/1, which is strongly protective against this disease, is associated with significantly thicker retinas in the perifovea. When controlling for age, Chr10-risk eyes with early or intermediate AMD have thinner retinas as compared to eyes from the Chr1-risk group with similar disease severity. Our analysis indicates that this difference likely results from distinct biological and disease initiation and progression events associated with Chr1- and Chr10-directed AMD.

Age-related macular degeneration (AMD) is the most common cause of central vision loss among elderly populations in industrialized countries. Genome-wide association studies have consistently associated two genomic loci with progression to late-stage AMD: the complement factor H (CFH) locus on chromosome 1q31 and the age-related maculopathy susceptibility 2-HtrA serine peptidase 1 (ARMS2-HTRA1) locus on chromosome 10q26. While the CFH risk variant has been shown to alter complement activity, the ARMS2-HTRA1 risk haplotype remains enigmatic due to high linkage disequilibrium and inconsistent functional findings spanning two genes that are plausibly causative for AMD risk. In this review, we detail the genetic and functional evidence used to support either ARMS2 or HTRA1 as the causal gene for AMD risk, emphasizing both the historical development and the current understanding of the ARMS2-HTRA1 locus in AMD pathogenesis. We conclude by summarizing the evidence in favor of HTRA1 and present our hypothesis whereby HTRA1-derived ECM fragments mediate AMD pathogenesis.

Optic neuritis (ON) and multiple sclerosis (MS) are complex diseases with multifactorial pathogenesis. The role of genetic factors in the development of these diseases is hypothesized, and specific biochemical components involved in the pathogenesis of ON and MS are yet to be determined. The aim of our study was to determine the associations between IL1RAP rs4624606, IL1RL1 rs1041973, IL-6 rs1800795, and HTRA1 rs11200638 gene polymorphisms and development of ON with or without MS.The study subjects included 80 ON patients and 146 healthy controls (HCs). Genotyping of IL1RAP rs4624606, IL1RL1 rs1041973, IL-6 rs1800795, and HTRA1 rs11200638 was performed using real-time polymerase chain reaction.A/C genotype of IL1RL1 rs1041973 was more frequent in ON patients than in HC subjects (Our study showed that IL1RAP rs4624606, IL-6 rs1800795, and HTRA1 rs11200638 are not associated with an increased risk of developing ON. However, the IL1RL1 rs1041973 A/C genotype might be associated with an increased risk of developing ON.

HtrA proteases regulate cellular homeostasis and cell death. Their dysfunctions have been correlated with oncogenesis and response to therapeutic treatment. We investigated the relation between HtrA1-3 expression and clinicopathological, and survival data, as well as the microsatellite status of tumors. Sixty-five colorectal cancer patients were included in the study. The expression of HTRA1-3 was estimated at the mRNA and protein levels by quantitative PCR and immunoblotting. Microsatellite status was determined by high-resolution-melting PCR. We found that the HTRA1 mRNA level was higher in colorectal cancer tissue as compared to the unchanged mucosa, specifically in primary lesions of metastasizing cancer. The levels of HtrA1 and HtrA2 proteins were reduced in tumor tissue when compared to unchanged mucosa, specifically in primary lesions of metastasizing disease. Moreover, a decrease in HTRA1 and HTRA2 transcripts&rsquo, levels in cancers with a high level of microsatellite instability compared to microsatellite stable ones has been observed. A low level of HtrA1 or/and HtrA2 in cancer tissue correlated with poorer patient survival. The expression of HTRA1 and HTRA2 changes during colorectal carcinogenesis and microsatellite instability may be, at least partially, associated with these changes. The alterations in the HTRA1/2 genes&rsquo, expression are connected with metastatic potential of colorectal cancer and may affect patient survival.

Background Cerebral autosomal recessive arteriopathy with subcortical infarcts and leukoencephalopathy (CARASIL) is an extremely rare monogenic autosomal disease associated with the HtrA serine protease 1 (HTRA 1) gene mutation. Recently, a few genetically confirmed CARASIL cases with novel HTRA1 mutations have been reported in countries other than Japan. Case Description Here, we report a case of a patient presenting with worsening right hemiplegia and hemiparesthesia. Physical examination revealed that the patient had typical clinical features of CARASIL including thinning hair, cognitive impairment, emotional changes, lumbago, and gait disorder. Brain magnetic resonance imaging showed abnormal diffuse symmetric changes in white matter and hypertensive diffusion-weighted imaging signals in the left centrum ovale and right splenium of the corpus callosum, and susceptibility-weighted imaging showed multiple cerebral microbleeds. Lumbar magnetic resonance imaging showed herniated disks with degenerative changes. A genetic test showed a novel homozygous nucleotide variation of c.847G>T in the HTRA1 gene, thereby resulting in p.Gly283Ter. Thus the patient met the diagnostic criteria for CARASIL. We provide a literature review of genetically confirmed CARASIL cases reported to date. Conclusions CARASIL is a rare autosomal recessive disease with an HTRA1 mutation. Familiarity with the early clinical and imaging features of CARASIL combined with a genetic test is key for its early diagnosis.

High temperature requirement A1 (HtrA1) is a serine protease detected in maternal plasma and in placental tissues during normal gestation and in various pathological conditions. The purpose of this study was to determine whether the maternal plasma concentration of HtrA1 in first trimester, alone or combined with other maternal factors, can be used to identify women at risk for spontaneous preterm birth (SPTB).This is a cohort study on pregnant women at 12 weeks of gestation recruited between 2014 and 2016 and prospectively followed until delivery. One hundred and fifty-nine women were included in the study: 140 women delivered at term and 19 (11.9%) delivered spontaneously preterm. Plasma samples were assessed for HtrA1 by ELISA and data were compared between women which delivered at term with women which delivered preterm. A multiple logistic regression analysis was used to estimate the independent effect of women's characteristics on the probability of a SPTB.SPTB was significantly associated with log HtrA1 values at 12 weeks of gestation, BMI before pregnancy and physical activity. In particular, the probability of a SPTB increases of 79% for every added unit of log HtrA1, while decreases of 18% for every added unit of BMI. In addition, physical activity was found as an important protective factor. The ROC curve showed that the model had a good accuracy in predicting SPTB, with an AUC equal to 0.83 (95%CI: 0.73-0.91).Maternal plasma HtrA1 may be considered a marker of SPTB. In addition, our model indicates two factors that could be modified to reduce the risk of SPTB, i.e. BMI before pregnancy and maternal physical activity.

Genetic studies have investigated the pathogenesis of age-related macular degeneration (AMD). The pachychoroid concept has recently garnered attention as a possible explanation for AMD pathogenesis; the genetic characteristics of pachychoroid diseases have also been elucidated. In this review, we summarize previously reported genetic characteristics of AMD and pachychoroid diseases, and analyze these data to understand the pathogenesis of AMD and pachychoroid diseases. Previous studies show that VIPR2 and the CFH I62V A allele promote development of pachychoroid and central serous chorioretinopathy (CSC), while the CFH I62V G allele promotes development of drusen, pachychoroid neovasculopathy (PCN/PNV), and AMD. ARMS2/HTRA1 also promotes development of drusen, PCN/PNV, and AMD. TNFRSF10A and GATA5 are associated with CSC but not with pachychoroid, and TNFRSF10A is associated with AMD that includes PCN/PNV. These genetic characteristics suggest the following mechanisms of developing AMD and pachychoroid diseases. VIPR2 and the CFH I62V A allele promote pachychoroid development, which can result in CSC development. The CFH I62V G allele promotes a common step during PCN/PNV and AMD development induced by pachychoroid or drusen, such as damage of Bruch's membrane or retinal pigment epithelium (RPE). ARMS2/HTRA1 also promotes damage of Bruch's membrane or RPE, while the association with drusen formation is stronger in ARMS2/HTRA1 than in CFH. TNFRSF10A and GATA5 promote blood-retinal-barrier breakdown to induce CSC, which could lead to PCN/PNV development. Furthermore, recently reported genetic associations with the natural course of CSC suggest the importance of reconsidering the subtype classification of CSC. These associations would enable the development of personalized/precision medicine for CSC and.

Startling reports described the paradoxical triggering of the human mitogen-activated protein kinase pathway when a small-molecule inhibitor specifically inactivates the BRAF V600E protein kinase but not wt-BRAF. We performed a conceptual analysis of the general phenomenon "activation by inhibition" using bacterial and human HtrA proteases as models. Our data suggest a clear explanation that is based on the classic biochemical principles of allostery and cooperativity. Although substoichiometric occupancy of inhibitor binding sites results in partial inhibition, this effect is overrun by a concomitant activation of unliganded binding sites. Therefore, when an inhibitor of a cooperative enzyme does not reach saturating levels, a common scenario during drug administration, it may cause the contrary of the desired effect. The implications for drug development are discussed.

Background Advanced age-related macular degeneration (AMD) is a leading cause of blindness. While around half of the genetic contribution to advanced AMD has been uncovered, little is known about the genetic architecture of early AMD. Methods To identify genetic factors for early AMD, we conducted a genome-wide association study (GWAS) meta-analysis (14,034 cases, 91,214 controls, 11 sources of data including the International AMD Genomics Consortium, IAMDGC, and UK Biobank, UKBB). We ascertained early AMD via color fundus photographs by manual grading for 10 sources and via an automated machine learning approach for > 170,000 photographs from UKBB. We searched for early AMD loci via GWAS and via a candidate approach based on 14 previously suggested early AMD variants. Results Altogether, we identified 10 independent loci with statistical significance for early AMD: (i) 8 from our GWAS with genome-wide significance (P − 8), (ii) one previously suggested locus with experiment-wise significance (P ARMS2/HTRA1, CFH, C2, C3, CETP, TNFRSF10A, VEGFA, APOE), except two that have not yet been identified with statistical significance for any AMD. Among the 17 genes within these two loci, in-silico functional annotation suggested CD46 and TYR as the most likely responsible genes. Presence or absence of an early AMD effect distinguished the known pathways of advanced AMD genetics (complement/lipid pathways versus extracellular matrix metabolism). Conclusions Our GWAS on early AMD identified novel loci, highlighted shared and distinct genetics between early and advanced AMD and provides insights into AMD etiology. Our data provide a resource comparable in size to the existing IAMDGC data on advanced AMD genetics enabling a joint view. The biological relevance of this joint view is underscored by the ability of early AMD effects to differentiate the major pathways for advanced AMD.

Vascular smooth muscle cell (VSMC) dysfunction is a hallmark of small vessel disease, a common cause of stroke and dementia. Two of the most frequently mutated genes in familial small vessel disease are HTRA1 and NOTCH3. The protease HTRA1 cleaves the NOTCH3 ligand JAG1 implying a mechanistic link between HTRA1 and Notch signaling. Here we report that HTRA1 is essential for VSMC differentiation into the contractile phenotype. Mechanistically, loss of HTRA1 increased JAG1 protein levels and NOTCH3 signaling activity in VSMC. In addition, the loss of HTRA1 enhanced TGFβ-SMAD2/3 signaling activity. Activation of either NOTCH3 or TGFβ signaling resulted in increased transcription of the HES and HEY transcriptional repressors and promoted the contractile VSMC phenotype. However, their combined over-activation led to an additive accumulation of HES and HEY proteins, which repressed the expression of contractile VSMC marker genes. As a result, VSMC adopted an immature phenotype with impaired arterial vasoconstriction in Htra1-deficient mice. These data demonstrate an essential role of HTRA1 in vascular maturation and homeostasis by controlling Notch and TGFβ signaling.

The HtrA protein family is composed by evolutionally-conserved serine proteases, which are homologous to the HtrA protein of the model bacterium Escherichia coli. They are widely distributed in organisms including humans, prokaryotes and eukaryotes. Moreover, HtrA family proteins are important regulators of a variety of human physiological processes, which contains the maintenance of mitochondrial homeostasis, cellular signal transduction and apoptosis regulation. The HtrA family has been found to be associated with cancer and could be used as a target for future cancer treatments. The purpose of this article is to review the relationship between these HtrA and cancer and to summarize the latest researches on HtrA and cancer.

TGFBI-associated corneal dystrophies are inherited disorders caused by TGFBI gene variants that promote deposition of mutant protein (TGFBIp) as insoluble aggregates in the cornea. Depending on the type and position of amino acid substitution, the aggregates may be amyloid fibrillar, amorphous globular or both, but the molecular mechanisms that drive these different patterns of aggregation are not fully understood. In the current study, we report the protein composition of amyloid corneal aggregates from lattice corneal dystrophy patients of Asian origin with H626R and R124C mutation and compared it with healthy corneal tissues via LC-MS/MS. We identified several amyloidogenic, nonfibrillar amyloid associated proteins and TGFBIp as the major components of the deposits. Our data indicates that apolipoprotein A-IV, apolipoprotein E, and serine protease HTRA1 were significantly enriched in patient deposits compared to healthy controls. HTRA1 was also found to be 7-fold enriched in the amyloid deposits of patients compared to the controls. Peptides sequences (G

Age-related macular degeneration (AMD) is a leading cause of visual impairment and irreversible blindness in most developed countries, affecting about 50 million elderly people worldwide. Retinal pigment epithelial (RPE) cell degeneration is the pathophysiological cause of AMD, leading to geographic atrophy and choroidal neovascularization. We and others have previously identified several polymorphisms on chromosome 10q26 (HTRA1 rs11200638 as well as LOC387715 rs10490924 and c.372_815del443ins54) associated with AMD. In this study, we confirmed the association of our previously identified HTRA1 insertion–deletion (indel) variant (c.34delCinsTCCT) in 195 exudative AMD patients and 390 controls from the Hong Kong Chinese cohort with additional 168 patients and 210 controls from the Chengdu Chinese cohort and followed by studying its biological functions in RPE cells. Genetic analysis verified the higher prevalence of c.34delCinsTCCT allele in control subjects (8.0%) than in AMD patients (1.9%; P=7.87 × 10−5, odds ratio=0.229). This protective effect was validated as the haplotype of the c.34delCinsTCCT allele existed independent of the risk haplotype (P=1.17 × 10−5). In vitro studies showed that recombinant HTRA1 c.34delCinsTCCT variant protein was more localized in the endoplasmic reticulum of RPE cells compared with the wild-type protein, and its secretion was delayed. Moreover, ARPE-19 cells expressing HTRA1 c.34delCinsTCCT variant had higher cell viability, lower cell apoptosis and were less responsive to anoikis, supporting its protective role. We revealed a protective AMD-associated HTRA1 variant in Chinese populations and the biological role of HTRA1 in RPE cell degeneration, indicating its involvement in AMD pathogenesis.