The SERPINF1 gene encodes pigment epithelium-derived factor (PEDF), a member of the serpin superfamily with neurotrophic and antiangiogenic properties in the retina. We hypothesized that absence of PEDF would lead to increased stress-associated retinal degeneration in Serpinf1 null mice. Accordingly, using a Serpinf1 null mouse model, we investigated the impact of PEDF absence on retinal morphology, and susceptibility to induced and inherited retinal degeneration. We studied the pattern of Serpinf1 expression in the mouse retina layers. PEDF protein was detected by western blotting. Transmission electron microscopy was performed on mouse retina. Serpinf1 null mice and wild type littermates were injected with NaIO₃ (30 mg/kg body weight) intraperitonially. At post-injection day 1, 3, 4, 6 and 8 mice were euthanized, and eyes were enucleated. Serpinf1 null and rd10 double mutant mice were generated and their eyes enucleated at different time points from post-natal day 15 to post-natal day 28. Enucleated eyes were processed for hematoxylin and eosin staining and histopathological evaluations. We found that Serpinf1 was expressed in the retinal pigment epithelium, in the inner nuclear layer and in the ganglion cell layer, but undetectable in the outer nuclear layer of wild type mice. Plasma PEDF protein levels were undetectable in Serpinf1 null animals. RPE atrophy and retinal thinning were observed in NaIO₃-treated wild type mice that progressed with time post-injection. NaIO₃-treated Serpinf1 null mice showed comparatively better retinal morphology than wild type mice at day 4 post-injection. However, the absence of PEDF in Serpinf1 null x rd10 mice increased the susceptibility to retinal degeneration relative to that of rd10 mice. We concluded that histopathological evaluation of retinas lacking PEDF showed that removal of the Serpinf1 gene may activate PEDF-independent compensatory mechanisms to protect the retina against oxidative stress, while it increases the susceptibility to degenerate the retina in inherited retinal degeneration models.

Keywords: NaIO(3); PEDF; Pigment epithelium-derived factor; Retinal degeneration; Serpinf1; rd10.

We report on a newborn with IUGR, rhizomelic dwarfism, and suspected chondrodysplasia punctata. At birth, OI was suspected; however, a skeletal survey suggested ML II alpha/beta. Sequencing revealed compound heterozygosity for a reported pathogenic and novel but expected pathogenic GNPTAB variant. Molecular testing for autosomal recessive OI identified a SERPINF1 variant.

Background: Heritable disorders of connective tissue (HDCT) is a heterogeneous group of conditions caused by defects in genes responsible for extracellular matrix elements. Although next-generation sequencing (NGS) technology can be used to analyze many genes at a time, precisely diagnosing HDCT is still challenging because of the overlapping phenotypes and genotypes.

Methods: A 67-gene NGS targeted panel or whole-exome sequencing was employed for the diagnosis of HDCT at a single medical center over 4 years. The phenotypes and genotypes of patients were analyzed retrospectively.

Results: Mutations in 16 genes were discovered in 34 patients with the suspicion of Ehlers-Danlos syndrome (n=7), Marfan syndrome (n=2), osteogenesis imperfecta (n=3), skeletal dysplasia (n=18), and others (n=4). Eighteen patients were found to have mutations in collagen genes, three had SERPINF1 mutations, two had TRPV4 mutations, two had FBN1 mutations, two had COMP mutations, and mutations in seven other genes were found in one patient each. The eight patients with COL1A1 mutations had a wide variation in phenotype. Patients with COL3A1 and COL5A1 mutations presented with classic EDS, those with SERPINF1 mutations presented with typical OI type VI, those with TRPV4 mutations presented with severe spinal deformity, and those with COL2A1 mutations presented with syndromic or nonsyndromic bone dysplasia or only short stature.

Conclusion: A wide diversity in HDCT was observed at a single center. Therefore, knowledge about the phenotype-genotype correlation in HDCT is still crucial in the diagnosis of this group of diseases, and an improvement in the screening tool will be needed.

Keywords: genetic heterogeneity; heritable disorders of connective tissue; next-generation sequencing; orthopedics; panel.

Objective: To explore the genotypes and phenotypes of osteogenesis imperfecta (OI) in Xinjiang Uygur children. Methods: The history of nine Uygur children with OI who were hospitalized in First Affiliated Hospital of Xinjiang Medical University from January 2013 to December 2017 were retrospectively reviewed. They were classified into 4 types according to the classical Sillence classification. The genes associated with OI were detected, and the pathogenic variation was assessed by InterVar and Alamut software according to the American College of Medical Genetics and Genomics (ACMG) recommendations. The phenotypes of children with different genotypes were further analyzed. Results: Nine cases aged 3 years and 6 monthes to 15 years were all clinically diagnosed as OI, the clinical manifes tations were repeated fractures, skeletal deformities,short stature, blue sclera, abnormol hearing, hypoplasia of dentin, and relaxation of Joint ligaments, among whom 6 was type Ⅲ OI, 3 were type Ⅳ OI. Nine mutations in 3 genes (COL1A1, COL1A2, and SERPINF1) were detected, and 5 of them were first reported and were all pathogenic variations. Conclusions: The cinical phenotypes of osteogenesis imperfecta in Xinjiang Uygur are complex and varied, but all of them have fractures and skeletal deformities. Genotype is different from that reported at China and abroad, and the SERPINF1 gene may have a higher incidence in Uyghur population. The genetic heterogeneity and unique gene variation pedigree of Uyghur osteogenesis imperfecta defects further provide a basis for the correlation between genotype and phenotype of osteogenesis defects.

Objectives Osteogenesis imperfecta type VI (OI VI) follows a progressive and severe course, yet unlike other forms of severe OI it has a later onset of fractures, and extra-skeletal findings are not part of the clinical picture. Another difference is that there is an increase in unmineralized osteoid tissue in OI VI, which hinders the effect of bisphosphonates-the current standard of treatment for OI. Therefore, the response to standard treatments in OI VI is not satisfactory. Herein, we report long-term follow-up of two cases with novel SERPINF1 mutations, who show great variation in their treatment response to bisphosphonates. Case presentation The first case was given pamidronate at the age of 15 months when he could sit independently, followed a fluctuating course under treatment, fracture rate did not decrease, however he was able to mobilize with walker at the age of 10 years. On the other hand, the second case developed severe deformities and became wheelchair-bound under pamidronate, thus the treatment was switched to denosumab. Unfortunately, there was no improvement under denosumab after 15 months too, and since bone pain increased, denosumab treatment was stopped. He was put on zoledronic acid instead. Conclusion SERPINF1 transcript amount may be an important factor to explain the variation in response to pamidronate therapy. In OI VI patients, the factors affecting the clinical course should be identified and new or combined treatment options should be established.

Keywords: OI type VI; PEDF; SERPINF1 mutation; genotype; osteogenesis imperfecta.

Osteogenesis imperfecta (OI) is a heterogeneous disorder that causes fragility, deformity, and fractures in bones. A large number of genes that are associated with the disease have been identified in the last decade; this makes the genetic diagnosis of OI more difficult. To improve our knowledge of the genetic mutation profile in OI we used single-stranded conformation polymorphism screening and automated sequencing to investigate the SERPINH1, FKBP10, and SERPINF1 genes, which are related to recessive OI, in 23 unrelated Brazilian patients. Nine rare changes and four common polymorphisms were detected. Most changes were benign genetic variants. In general, changes in the SERPINH1 and SERPINF1 genes were synonymous polymorphisms or missense changes located in non-coding regions. A pathogenic change was found in the FKBP10 gene. The characterization of mutations related to OI in distinct populations can improve our knowledge of the genetic aspects of OI and help us develop molecular strategies for the diagnosis of the disease.

BACKGROUND

Osteogenesis imperfecta (OI) is a rare genetic bone disease characterized by bone fragility and low bone mass. OI was mainly caused by genetic mutations in collagen genes, COL1A1 and COL1A2. Nevertheless, new genes have been identified to be causally linked to OI. The clinical features between each OI groups share great similarities and it is sometimes difficult for clinicians to diagnose the disease accurately. Here, we identify the genetic mutations of OI patients from Malaysia and correlate the genetic mutations with the clinical features.

METHODS

Targeted sequencing of fourteen genes panel was performed to identify the mutations in 29 OI patients with type I, III, IV and V disease. The mutations were determined using Ion Torrent Suite software version 5 and variant annotation was conducted using ANNOVAR. The identified mutations were confirmed using Sanger sequencing and in silico analysis was performed to evaluate the effects of the candidate mutations at protein level.

RESULTS

Majority of patients had mutations in collagen genes, 48% (n = 14) in COL1A1 and 14% (n = 4) in COL1A2. Type I OI was caused by quantitative mutations in COL1A1 whereas most of type III and IV were due to qualitative mutations in both of the collagen genes. Those with quantitative mutations had milder clinical severity compared to qualitative mutations in terms of dentinogenesis imperfecta (DI), bone deformity and the ability to walk with aid. Furthermore, a few patients (28%, n = 8) had mutations in IFITM5, BMP1, P3H1 and SERPINF1.

CONCLUSIONS

Majority of our OI patients have mutations in collagen genes, similar to other OI populations worldwide. Genotype-phenotype analysis revealed that qualitative mutations had more severe clinical characteristics compared to quantitative mutations. It is crucial to identify the causative mutations and the clinical severity of OI patients may be predicted based on the types of mutations.

Osteogenesis imperfecta (OI) Type VI is characterized by a defect in bone mineralization, which results in multiple fractures early in life. Null mutations in the PEDF gene, Serpinf1, are the cause of OI VI. Whether PEDF restoration in a murine model of OI Type VI could improve bone mass and function was previously unknown. In Belinsky et al, we provided evidence that PEDF delivery enhanced bone mass and improved parameters of bone function in vivo. Further, we demonstrated that PEDF temporally inhibits Wnt signaling to enhance osteoblast differentiation. Here, we demonstrate that generation of induced pluripotent stem cells (iPSCs) from a PEDF null patient provides additional evidence for PEDF's role in regulating extracellular matrix proteins secreted from osteoblasts. PEDF null iPSCs have marked abnormalities in secreted matrix proteins, capturing a key feature of human OI Type VI, which were normalized by exogenous PEDF. Lastly, we place our recent findings within the broader context of PEDF biology and the developmental signaling pathways that are implicated in its actions.

Mutations in several genes, including SERPRINF1 and COL1A1, have been associated with the development of osteogenesis imperfecta (OI). Here, we reported the co-occurrence of a rare heterozygous variant (c.167C>G p.Ala56Gly) in SERPRINF1 and a novel heterozygous mutation (c.1634G>A p.Gly545Asp) in COL1A1 in a foetus with a severe form of OI. Bioinformatics modelling revealed that the effect of the mutation on SPERINF1 is neutral. In contrast, the mutation in COL1A1 is deleterious. It is predicted to cause distortion of the α (1) chain of the type I collagen and results in structural instability of the protein. Therefore, a novel dominant variant of COL1A1 likely underlies the severe foetal pathology observed, although we do not exclude the possibility that the heterozygous mutations in SERPINF and COL1A1 may interact and co-ordinately cause pathogenesis. This novel COL1A1 mutation is recommended to be included in the diagnostic panels for OI.

Objective: The authors aim to provide genetic counselling and prenatal gene diagnosis to the families with osteogenesis imperfecta(OI), based on the identification of pathogenetic mutations in large cohort genetic testing. Methods: DNA was extracted from the peripheral blood of parents of the fetuses, and from the villi tissue, amniotic fluid or cord blood of the fetuses using a standard sodium dodecyl sulfate-proteinase K-phenol/chloroform extraction method. PCR combined with Sanger DNA sequencing was performed to validate the pathogenic mutations of 200 fetuses at risk of OI and their parents from 158 families. Allelic analysis of microsatellite markers was applied to exclude the false positive caused by maternal DNA contamination, when both the fetus and the mother harbored the same pathogenic genotype. Results: A total of 83 affected fetuses (83/200, 41.5%) and 12 (12/200, 6.0%) recessive carriers were identified among the 200 fetuses. The 83 affected fetuses included 78 heterozygotes (45 of COL1A1, 32 of COL1A2, one of IFITM5), and 5 compound heterozygotes or homozygotes of recessive OI (two of FKBP10, one of SEC24D, one of WNT1 and one of CRTAP); The 12 recessive carriers included 7 of WNT1, 4 of SERPINF1 and one of SERPINH1. Maternal DNA contamination was excluded from the genomic DNA samples of OI fetuses when their mother with the same affected genotypes. Conclusion: In this study, the authors used an optimized gene diagnosis system of OI to perform prenatal genetic diagnosis to 200 fetuses at high risk of OI, and provided precisely genetic counselling to the OI families.

OBJECTIVE

To investigate the relationship between genotype and severity of malocclusion in osteogenesis imperfecta (OI).

METHODS

A total of 49 patients participated in this cross-sectional study (age range: 5-19 years; 28 females; diagnoses: OI type I, N = 7; OI type III, N = 11; OI type IV, N = 27; OI type V, N = 2; OI type VI, N = 2).

METHODS

Sequence analysis of COL1A1/COL1A2 and other OI-related genes was compared to the Peer Assessment Rating (PAR), an index reflecting the severity of malocclusion.

RESULTS

The mutation spectrum was as follows: COL1A1, N = 22; COL1A2, N = 22, IFITM5, N = 2; SERPINF1, N = 2; no mutation detected, N = 1). Compared to patients with COL1A1 mutations, patients with COL1A2 mutations had significantly higher scores for total PAR, anterior cross-bite, anterior open bite and anteroposterior buccal occlusion. Males with COL1A2 mutations had significantly higher total PAR scores than females (median 36 vs 30, P = .047, Mann-Whitney test). Exploratory correlation between age and buccal vertical occlusion was noted in patients with COL1A2 mutations (Spearman correlation: r = .46, P = .03, power = .50). Two patients with OI type V (caused by IFITM5 mutations) had total PAR scores of 44 and 21. Both patients scored high for "segment." Patients with OI type VI (due to SERPINF1 mutations) scored similar to OI type V for "centreline." Considerable difference was observed in the total PAR score between the 2 patients with OI type VI. They had total PAR of 43 and 2.

CONCLUSIONS

Type of disease-causing mutation affects the severity of malocclusion in individuals with OI.

Sprouting angiogenesis is the formation of new capillaries from existing vessels in response to tissue hypoxia due to growth/development, repair/healing, and also chronic inflammation. In this study, we aimed to elucidate the effect of IL-6, a pleiotropic cytokine with both pro-inflammatory and anti-inflammatory functions, in regulating the sprouting angiogenic response of endothelial cells (ECs). We found that activation of IL-6 trans-signaling inhibited the migration, proliferation, and tube formation ability of ECs. In addition, inhibition of the autocrine IL-6 classic-signaling by depleting endogenous IL-6 from ECs impaired their tube formation ability. At the molecular level, we found that IL-6 trans-signaling in ECs upregulated established endogenous anti-angiogenic factors such as CXCL10 and SERPINF1 while at the same time downregulated known endogenous pro-angiogenic factors such as cKIT and CXCL8. Furthermore, prior activation of ECs by IL-6 trans-signaling alters their response to vascular endothelial growth factor-A (VEGF-A), causing an increased p38, but decreased Erk1/2 phosphorylation. Collectively, our data demonstrated the dual facets of IL-6 in regulating the sprouting angiogenic function of ECs. In addition, we shed light on molecular mechanisms behind the IL-6 trans-signaling mediated impairment of endothelial sprouting angiogenic response.

Keywords: HUVECs; IPA; Matrigel; VEGF-A signaling; cytokine; neoangiogenesis.

BACKGROUND

SERPINF1 mutations caused deficiency of pigment epithelium-derived factor (PEDF) and would lead to osteogenesis imperfecta (OI) type VI. However, serum PEDF levels were unclear in Chinese OI patients who had clear molecular diagnosis.

OBJECTIVE

To assess PEDF levels in different genotypes of OI, to evaluate the influencing factors of PEDF in Chinese OI patients with clear molecular diagnosis.

METHODS

Known candidate genes of OI were examined by a targeted next generation sequence. Serum PEDF levels were measured by ELISA in 6 OI patients with SERPINF1 mutations, 6 carriers of one copy of the SERPINF1 mutation, 88 OI patients with COL1A1, CLO1A2, IFITM5 and other pathogenic mutations of OI and 24 healthy controls. We compared the differences in serum PEDF levels among different OI patients and normal controls.

RESULTS

Serum PEDF levels were extremely low in OI patients with SERPINF1 mutations (0.66±1.60μg/ml) than in OI patients with other pathogenic mutations (4.88±1.43-7.07±2.43μg/ml), carriers of one copy of SERPINF1 mutation (4.94±2.35μg/ml), and normal controls (7.29±2.31μg/m) (P<0.001). No significant differences in serum PEDF concentrations were found among patients with OI type I, III or IV, and between patients with or without bisphosphonate treatment. Serum PEDF level was positively correlated with Z-score of weight (r=0.310, P=0.004), BMI (r=0.253, P=0.020) and alanine aminotransferase (r=0.291, P=0.007).

CONCLUSIONS

Extremely low level of PEDF was demonstrated as a specific, convenient, and inexpensive diagnostic biomarker for OI patients with SERPINF1 mutations, but it could not provide information regarding the clinical severity of OI and the efficacy of bisphosphonates treatment.

Background: Hypertension (HTN) patients who have phlegm-dampness syndrome (PDS) tend to be obese and have worse outcomes. However, the association of body weight (BW) changes and mechanisms underlying the pathophysiology of HTN-PDS are not well elucidated. This study aims to identify the longitudinal observations associated with the circulating markers discriminating BW changes of individuals with HTN-PDS.

Methods: An integrative approach relying on metabolomics and proteomics was applied to serum samples from HTN-PDS patients in a prospective cohort to identify the plausible mechanistic pathways underpinning HTN-PDS pathophysiology. Study participants were determined to have experienced a weight change if they showed a 5%-15% increase/reduction in BW at the end of the follow-up period. The joint pathway analysis and network analysis were performed using Ingenuity Pathway Analysis (IPA®) on the serum samples obtained from the participants over the period.

Results: The study involved 22 HTN-PDS patients who were overweight initially and were able to lose enough weight and 24 HTN-PDS individuals who developed overweight from normal BMI during a one-year follow-up. Our analysis suggested three types of phosphatidylcholine (PC) were altered. PC (22:2(13Z,16Z)/24:1(15Z)) and LysoPC (16:1(9Z)) were decreased in Queryweight gain samples, whereas the levels of PC (14:0/16:0) were increased in weight loss samples. The metabolomic analysis suggested 24 metabolites associated with HTN-PDS. Of them, 13 were up-regulated and 11 were down-regulated. The two-dimensional difference gel electrophoresis (2D DIGE) identified 45 phosphorylated proteins got altered in the HTN-PDS patients, wherein 23 were up-regulated and 22 were down-regulated. Integrated proteomic and metabolomics analyse acknowledged biomarkers PC, Complement C3, C4a/C4b, A2M and SERPINF1 as strong predictors for BW changes in HTN-PDS patients.

Conclusion: The combined serum proteomic and metabolomic profiling reveals a link between BW change and the complement system activity, altered phosphatidylcholine metabolism in HTN-PDS patients. Future studies with larger cohorts are required to strengthen and validate these findings.

Keywords: Chinese medicine syndrome; Hypertension; Longitudinal study; Metabolomics; Proteomics.

The occurrence of fractures in young individuals is frequently overlooked by physicians, especially when associated with exercise or trauma. Nevertheless, multiple fractures should always be investigated since underlying conditions can predispose to such events. We describe here the case of a young, healthy woman who sustained multiple fractures in the lower limbs, which were initially considered to be "stress fractures". Further investigation, including a panel of genes associated with osteogenesis imperfecta, revealed that the patient is a heterozygous carrier of a SERPINF1 variant. According to criteria recommended by the American College of Medical Genetics and Genomics and the Association for Molecular Pathology, this variant is classified as likely benign (PM2, PP3, PP4, BP1, and BP4). The patient's mother and brother were also asymptomatic carriers of the variant and had sustained previous minor fractures. The patient had normal biochemical profile and bone density. This condition has been rarely described and is not associated with low bone mineral density or altered bone turnover markers. This case highlights the importance of investigating multiple fractures in young patients who are otherwise healthy since these may be a warning sign of rare genetic conditions associated with fragility fractures.

Background/objectives: In England, children (0-18 years) with severe, complex and atypical osteogenesis imperfecta (OI) are managed by four centres (Birmingham, Bristol, London, Sheffield) in a 'Highly Specialised Service' (HSS OI); affected children with a genetic origin for their disease that is not in COL1A1 or COL1A2 form the majority of the 'atypical' group, which has set criteria for entry into the service. We have used the data from the service to assess the range and frequency of non-collagen pathogenic variants resulting in OI in a single country.

Methods: Children with atypical OI were identified through the HSS OI service database. All genetic testing for children with OI in the service were undertaken at the Sheffield Diagnostic Genetics Service. Variant data were extracted and matched to individual patients. This study was done as part of a service evaluation project registered with the Sheffield Children's Hospital Clinical Governance Department.

Results: One hundred of 337 children in the HSS met the 'atypical' criteria. Eighty have had genetic testing undertaken; 72 had genetic changes detected, 67 in 13 genes known to be causative for OI. The most frequently affected genes were IFITM5 (22), P3H1 (12), SERPINF1 (8) and BMP1 (6).

Conclusion: Among children with more severe forms of OI (approximately one-third of all children with OI), around 20% have pathogenic variants in non-collagen genes. IFITM5 was the most commonly affected gene, followed by genes within the P3H1 complex. These data provide additional information regarding the likelihood of different genetic origins of the disease in children with OI, which may influence clinical care.

Keywords: child health; epidemiology; genetics; paediatrics.

Toll-like receptors (TLR) 4 and its endogenous ligands are highly expressed in aorta. In the present study, we have explored the effect of TLR-4 activation by pro-inflammatory and angiogenic factors in PBMCs of patients with Takayasu Arteritis (TA). In the screening cohort, PBMCs of TA (n = 6) and healthy controls (n = 6) were stimulated with LPS and cultured. mRNA expression of 84 genes were quantitated by RT2 Profiler™ PCR Array kit in PBMCs. Validation set of additional PBMCs from TA (n = 7) and healthy controls [HC) (n = 7) were then stimulated with LPS to study expression of selected genes with delta Ct > 0.1 in the screening cohort. Significant gene expressions were correlated with Indian Takayasu arteritis activity scores (ITAS 2010). Increased expression of CCL2 was observed only in unstimulated PBMCs of patients with TA [median relative difference (RD) of 2.37] as compared to HC (RD 1.37, p < 0.03) in validation cohort, while stimulation with TLR4 ligand led to increased mRNA expression of IL-1β (RD 7.9, p < 0.028) and IL-1R2 (RD 0.08 p < 0.013) genes as compared to that of HC [RD of 5.32 for IL-1β and 0.01 for IL-1R2, respectively] in validation cohort. TLR4 activation also led to significantly higher expression of HPSE, TIMP1 and low expression of VEGFB, S1PR1, SERPINF1, ANGPLT4, ANGPT2, TIE1 and NOS3 genes in the screening cohort. But expression of VEGFB was not significant in validation cohort. The significant gene expressions, however, did not correlate with ITAS [ITAS2010 and ITAS-A (CRP)]. TLR4 activation leads to increased expression of IL-1β and IL-1R2 genes in PBMCs of patients with TA.

Keywords: Angiogenesis; Autoinflammatory disease; India; Takayasu arteritis; Toll-like receptor 4.

OBJECTIVE

To detect the expressions of receptor tyrosine kinases (RTKs) mRNA and protein and to explore potentially promising tumor markers and conceivable drug target in bladder cancer.

METHODS

The expressions of RTKs mRNA and protein in tissue from invasive urothelial carcinoma of the bladder were examined by real-time quantitative PCR array and cytokine antibody array, with normal bladder tissue as control. The Results were analyzed using bioinformatic approaches.

RESULTS

The expressions of TGFA, STAB1, SERPINE1, ANGPT2, SPINK5, ANGPTL1, PROK1, MDK, CXCL9, GRN, RUNX1, VEGFA, and TGFB1 were obviously upregulated in bladder cancer tissue, while those of EDIL3, PTN, CCL2, PDGFD, FGF13, KITLG, FGF2, SERPINF1, and TNF were downregulated. ALK, Btk, EphB2, ErbB4, PDGFR-α, ROS, Tie-2, Tyk2, and VEGFR3 were over-expressed in bladder cancer, while FRK, Fyn, IGF-IR, Insulin R, Itk, JAK1, JAK3, and LCK were low-expressed.

CONCLUSIONS

Vascular endothelial growth factor/platelet-derived growth factor-targeted therapies may play an active role in treating carcinoma of bladder.

Pigment epithelium-derived factor (PEDF, also known as SERPINF1) is a secreted glycoprotein with neuroprotective effects. However, the potential role of PEDF in major depressive disorder (MDD) remains largely unknown. Here, applying two-dimensional gel electrophoresis (2-DE) proteomics, we found that PEDF levels were significantly decreased in the plasma of 12 first-episode treatment-naïve MDD patients (FETND) compared to the levels in 12 healthy controls (HCs). PEDF levels were especially lower in MDD patients than in HCs and patients with bipolar disorder (BD) and schizophrenia (SCZ), and elevated PEDF were consistent with decreased HAM-D scores in patients given antidepressant therapy (ADT). Animal research indicated that PEDF was decreased in the periphery and hippocampus of two well-known depression rodent models (the chronic unpredictable mild stress (CUMS) rat model and chronic social defeat stress (CSDS) mouse model). Decreased PEDF levels in the hippocampus led to depressive-like behaviors, synaptic impairments and aberrant Wnt signaling in C57BL mice, while increased PEDF resulted in the opposite results. Mechanistic studies indicated that PEDF contributes to dendritic growth and Wnt signaling activation in the hippocampus of adult mice. Taken together, the results of our study demonstrate the involvement of PEDF and its related mechanism in depression, thus providing translational evidence suggesting that PEDF may be a novel therapeutic target for depression.

Background: Osteogenesis imperfecta (OI) is a clinically and genetically heterogeneous group of diseases characterized by increased bone fragility and deformities. Although most patients with OI have heterozygous mutations in COL1A1 or COL1A2, 17 genes have been reported to cause OI, most of which are autosomal recessive (AR) inherited, during the last years. The aim of this study is to determine the mutation spectrum in Turkish OI cohort and to investigate the genotype-phenotype correlation.

Methods: 150 patients from 140 Turkish families with OI phenotype were included in this study. Mutations in OI-related genes were identified using targeted gene panel, MLPA analysis for COL1A1 and whole exome sequencing. 113 patients who had OI disease-causing variants were followed for 1-20 years.

Results: OI disease-causing variants were detected in 117 families, of which 62.4% in COL1A1/A2, 35.9% in AR-related genes. A heterozygous variant in IFITM5 and a hemizygous in MBTPS2 were also described, one in each patient. Eighteen biallelic variants (13 novel) were identified in nine genes (FKBP10, P3H1, SERPINF1, TMEM38B, WNT1, BMP1, CRTAP, FAM46A, MESD) among which FKBP10, P3H1 and SERPINF1 were most common. The most severe phenotypes were in patients with FKBP10, SERPINF1, CRTAP, FAM46A and MESD variants. P3H1 patients had moderate, while BMP1 had the mild phenotype. Clinical phenotypes were variable in patients with WNT1 and TMEM38B mutations. We also found mutations in ten genes (PLS3, LRP5, ANO5, SLC34A1, EFEMP2, PRDM5, GORAB, OCRL1, TNFRSF11B, DPH1) associated with diseases presenting clinical features which overlap OI, in eleven families.

Conclusion: We identified disease-causing mutations in 83.6% in a large Turkish pediatric OI cohort. 40 novel variants were described. Clinical features and long-term follow-up findings of AR inherited OI types and especially very rare biallelic variants were presented for the first time. Unlike previously reported studies, the mutations that we found in P3H1 were all missense, causing a moderate phenotype.

Keywords: Autosomal recessive; COL1A1; COL1A2; MLPA; Osteogenesis imperfecta; Whole exome sequencing.

Purpose: NK-5962 is a key component of photoelectric dye-coupled polyethylene film, designated Okayama University type-retinal prosthesis (OUReP™). Previously, we found that NK-5962 solution could reduce the number of apoptotic photoreceptors in the eyes of the Royal College of Surgeons (RCS) rats by intravitreal injection under a 12 h light/dark cycle. This study aimed to explore possible molecular mechanisms underlying the anti-apoptotic effect of NK-5962 in the retina of RCS rats.

Methods: RCS rats received intravitreal injections of NK-5962 solution in the left eye at the age of 3 and 4 weeks, before the age of 5 weeks when the speed in the apoptotic degeneration of photoreceptors reaches its peak. The vehicle-treated right eyes served as controls. All rats were housed under a 12 h light/dark cycle, and the retinas were dissected out at the age of 5 weeks for RNA sequence (RNA-seq) analysis. For the functional annotation of differentially expressed genes (DEGs), the Metascape and DAVID databases were used.

Results: In total, 55 up-regulated DEGs, and one down-regulated gene (LYVE1) were found to be common among samples treated with NK-5962. These DEGs were analyzed using Gene Ontology (GO) term enrichment, Kyoto Encyclopedia of Genes and Genomes (KEGG), and Reactome pathway analyses. We focused on the up-regulated DEGs that were enriched in extracellular matrix organization, extracellular exosome, and PI3K-Akt signaling pathways. These terms and pathways may relate to mechanisms to protect photoreceptor cells. Moreover, our analyses suggest that SERPINF1, which encodes pigment epithelium-derived factor (PEDF), is one of the key regulatory genes involved in the anti-apoptotic effect of NK-5962 in RCS rat retinas.

Conclusions: Our findings suggest that photoelectric dye NK-5962 may delay apoptotic death of photoreceptor cells in RCS rats by up-regulating genes related to extracellular matrix organization, extracellular exosome, and PI3K-Akt signaling pathways. Overall, our RNA-seq and bioinformatics analyses provide insights in the transcriptome responses in the dystrophic RCS rat retinas that were induced by NK-5962 intravitreal injection and offer potential target genes for developing new therapeutic strategies for patients with retinitis pigmentosa.

Keywords: PI3K–Akt signaling pathway; SERPINF1; apoptosis; drug; extracellular exosome; extracellular matrix organization; photoreceptors; pigment epithelium-derived factor (PEDF); retina; retinitis pigmentosa.

Pigment epithelium-derived factor (PEDF) is a multifunctional glycoprotein encoded by SERPINF1 and our previous study reported that PEDF may have antidepressant effects. As a key brain region regulating cognition, memory and emotion, the prefrontal cortex (PFC) has been studied extensively in major depressive disorder (MDD), but there are few reports on the relationship between PEDF and the PFC. In this study, enzyme-linked immunosorbent assay showed that the PEDF level was decreased in the plasma of MDD patients compared with that of healthy controls. Western blotting validated that the PEDF expression in the PFC was downregulated in the mouse chronic social defeat stress and rat chronic unpredictable mild stress models of depression. Correspondingly, normal mice overexpressing PEDF in the PFC showed depression-resistant phenotypes. We detected PFC metabolite levels by liquid chromatography-tandem mass spectrometry and found significant upregulation of 5-hydroxyindoleacetic acid, kynurenine, 5-hydroxytryptamine, ornithine and glutamine, and downregulation of 5-hydroxytryptophan, glutamic acid and aspartic acid in PEDF-overexpressing mice compared with control mice, in which no such changes were detected. Combined with the above findings, this provides an insight into a potential mechanism of the antidepressant effects of PEDF via the PFC, which may help to improve understanding of depression pathophysiology.

Keywords: Depression; Metabolomics; PEDF; Prefrontal Cortex.

Urine proteomic applications in children suggested their potential in discriminating between healthy subjects from those with respiratory diseases. The aim of the current study was to combine protein fractionation, by urinary extracellular vesicle isolation, and proteomics analysis in order to establish whether different patterns of respiratory impedance in healthy preschoolers can be characterized from a protein fingerprint. Twenty-one 3-5-yr-old healthy children, representative of 66 recruited subjects, were selected: 12 late preterm (LP) and 9 full-term (T) born. Children underwent measurement of respiratory impedance through Forced Oscillation Technique (FOT) and no significant differences between LP and T were found. Unbiased clustering, based on proteomic signatures, stratified three groups of children (A, B, C) with significantly different patterns of respiratory impedance, which was slightly worse in group A than in groups B and C. Six proteins (Tripeptidyl peptidase I (TPP1), Cubilin (CUBN), SerpinA4, SerpinF1, Thy-1 membrane glycoprotein (THY1) and Angiopoietin-related protein 2 (ANGPTL2)) were identified in order to type the membership of subjects to the three groups. The differential levels of the six proteins in groups A, B and C suggest that proteomic-based profiles of urinary fractionated exosomes could represent a link between respiratory impedance and underlying biological profiles in healthy preschool children.

Keywords: extracellular vesicle; forced oscillation technique; preschooler healthy children; proteomics; urine fractionation.