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What is known and objective Previous studies based on small-sample clinical data proved that short-term use of hypoxia-inducible factor prolyl hydroxylase (HIF-PHD) inhibitors increased haemoglobin levels in anaemic patients with chronic kidney disease (CKD). However, these studies reached conflicting conclusions on iron parameters and adverse event profiles. Our meta-analysis aimed to evaluate the long-term efficacy and safety of HIF-PHD inhibitors in renal anaemia. Methods Randomized controlled trials comparing treatment with HIF-PHD inhibitors versus placebo or erythropoiesis-stimulating agents (ESAs) were thoroughly searched in the PubMed, Embase, Cochrane Library and international clinical trial registries. Meta-analysis was performed on main outcomes with random effects models. Results and discussion A total of 30 studies comprising 13,146 patients were included. The HIF-PHD inhibitors used included roxadustat, daprodustat, vadadustat, molidustat, desidustat and enarodustat. HIF-PHD inhibitors significantly increased haemoglobin levels in comparison with placebo [weighted mean difference (WMD) 1.53, 95% confidence interval (CI) 1.39 to 1.67] or ESAs (WMD 0.13, 95% CI 0.03 to 0.22). Hepcidin, ferritin and serum iron levels were decreased, while total iron binding capacity and transferrin levels were increased in the HIF-PHD inhibitor group versus those in placebo or ESAs group. Additionally, HIF-PHD inhibitors medication was associated with cholesterol-lowering effects. As for safety, the risk of serious adverse events in the HIF-PHD inhibitor group was increased in comparison with placebo group [risk ratio (RR) 1.07, 95% CI 1.01 to 1.13], but comparable to the ESAs group (RR 1.02, 95% CI 0.94 to 1.10). Compared with placebo, the agents increased the risk of diarrhoea (1.21, 1.00 to 1.47), nausea (1.46, 1.09 to 1.97), oedema peripheral (1.32, 1.01 to 1.59), hyperkalemia (1.27, 1.05 to 1.54) and hypertension (1.34, 1.02 to 1.76). Compared with ESAs, the drugs increased the risk of vomiting (1.30, 1.02 to 1.65), headache (1.27, 1.05 to 1.53) and thrombosis events (1.31, 1.05 to 1.63). What is new and conclusion HIF-PHD inhibitors treatment effectively increased haemoglobin levels and promoted iron utilization in anaemic patients with CKD, and they were well tolerated for long-term use. In order to avoid unfavourable effects of excessive iron consumption, it was appropriate to administer HIF-PHD inhibitors in combination with iron supplements for long-term treatment.

Purpose To identify the pathogenic gene of infantile nystagmus syndrome (INS) in three Chinese families and explore the potential pathogenic mechanism of FERM domain-containing 7 (FRMD7) mutations. Methods Genetic testing was performed via Sanger sequencing. Western blotting was used to analyze protein expression of FRMD7. Glutathione S-transferase pull-down and immunoprecipitation were conducted to investigate the proteins interacting with FRMD7. Rescue assays were performed in Caenorhabditis elegans to explore the potential role of FRMD7 in vivo. Results We recruited three Chinese families with X-linked INS and identified a duplication and two missense mutations in FRMD7: c.998dupA/p.His333Glnfs*2, c.580G>A/p.Ala194Thr, and c.973A>G/p.Arg325Gly (one in each family). Expression levels of three mutants were similar to that of wild-type FRMD7 in vitro. Interestingly, the mutant p.His333Glnfs*2 exhibited a predominantly nuclear location, whereas wild-type FRMD7 localized to the cytoplasm. In addition, we found FRMD7 to directly interact with the loop between transmembrane domains 3 and 4 of GABRA2, a type A gamma-aminobutyric acid (GABA) receptor (GABAARs) subunit critical for receptor transport and localization, whereas the mutants p.Ala194Thr and p.Arg325Gly exhibited decreased binding to GABRA2. In frm-3 (a nematode homologue of FRMD7) null C. elegans, we found that FRMD7 mutants exhibited a poor rescue effect on the defects of locomotion and fluorescence recovery after photobleaching of GABAARs. Conclusions Our findings identified three FRMD7 mutants in three Chinese families with X-linked INS and confirmed GABRA2 as a novel binding partner of FRMD7. These findings suggest that FRMD7 plays an important role by targeting GABAARs.

Porokeratosis is recognized as a heterogenously inherited epidermal keratinization disorder. Disseminated superficial actinic porokeratosis (DSAP) is considered to be the most common form of porokeratosis and is characterized by multiple, small keratotic lesions on sun-exposed areas of body. MVK has been reported to be the main candidate gene associated with DSAP. In this study, six sporadic cases of DSAP were clinically characterized. Direct DNA sequencing analysis of the whole coding regions of MVK detected three MVK missense mutations, and two were novel for DSAP: c.31C>T (P11S) and c.1004G>A (G335D). The three mutant MVK proteins were less stable than the wild type protein in different degrees. Mutation G335D also resulted in the misfolding of the ATP binding domain. This study extends the mutation spectrum of MVK. MVK protein stability and correct folding might be the molecular mechanism causing DASP. A 50% probability of detecting a MVK mutation in six DSAP sporadic cases indicated that the MVK gene was useful for clinical characterization, genetic counseling and prenatal diagnosis of DSAP.

Heat shock factor 4 (HSF4) is an important transcriptional factor that plays a vital role in lens development and differentiation, but the mechanism underlying the regulation of HSF4 is ambiguous. BCAS2 was reported to be an essential subunit of pre-mRNA splicing complex. Here, we identified BCAS2 as a novel HSF4 interacting partner. High expression of BCAS2 in the lens epithelium cells and the bow region of mouse lens was detected by immunohistochemistry. In human lens epithelial cells, BCAS2 negatively regulates HSF4 protein level and transcriptional activity, whereas in BCAS2 knockdown cells, HSF4 protein stability was increased significantly. We further demonstrated that the prolonged protein half-time of HSF4 in BCAS2 knockdown cells was due to reduced ubiquitination. Moreover, we have identified the lysine 206 of HSF4 as the key residue for ubiquitination. The HSF4-K206R mutant blocked the impact of BCAS2 on HSF4 protein stability. Taken together, we identified a pathway for HSF4 degradation through the ubiquitin-proteasome system, and a novel function for BCAS2 that may act as a negative regulatory factor for modulating HSF4 protein homeostasis.

The differentiation from constantly dividing epithelial cells into secondary fiber cells is a key step during lens development. Failure in this process, which requires cell proliferation inhibition and cell cycle exit, causes cataract formation. HSF4 (Heat Shock Transcription Factor 4) gene mutations may lead to both congenital and senile cataract. However, how HSF4 mutations induce cataract formation remains obscure. In this study, we demonstrate that HSF4 can suppress the proliferation of human lens epithelial cells (HLECs) by promoting G1/S arrest in a p53-dependent manner. In contrast, HSF4 with cataract causative mutations fail to cause cell cycle arrest and have no obvious effect on cell proliferation. We further identify that HSF4 recruits p53 in the nucleus and promotes its transcriptional activity, leading to the expression of its target gene p21 in HLECs. HSF4, but not its cataract-causing mutants, stabilizes p53 protein and inhibits its ubiquitin degradation. Our data reveal that HSF4 may work as a switch between lens epithelial cell proliferation and secondary fiber cell differentiation, a process which mainly depends on p53. Through demonstration of this novel downstream pathway of HSF4, our results help uncover the pathogenic mechanisms caused by HSF4 mutations.

Mutations in the ceramide kinase-like gene (CERKL) are associated with severe retinal degeneration. However, the exact function of the encoded protein (CERKL) remains unknown. Here we show that CERKL interacts with mitochondrial thioredoxin 2 (TRX2) and maintains TRX2 in the reduced redox state. Overexpression of CERKL protects cells from apoptosis under oxidative stress, whereas suppressing CERKL renders cells more sensitive to oxidative stress. In zebrafish, CERKL protein prominently locates in the outer segment and inner segment of the photoreceptor of the retina. Knockdown of CERKL in the zebrafish leads to an increase of retinal cell death, including cone and rod photoreceptor degeneration. Signs of oxidative damage to macromolecules were also detected in CERKL deficient zebrafish retina. Our results show that CERKL interacts with TRX2 and plays a novel key role in the regulation of the TRX2 antioxidant pathway and, for the first time, provides an explanation of how mutations in CERKL may lead to retinal cell death.

Disseminated superficial porokeratosis (DSP) is a rare keratinization disorder of the epidermis. It is characterized by keratotic lesions with an atrophic center encircled by a prominent peripheral ridge. We investigated the genetic basis of DSP in two five-generation Chinese families with members diagnosed with DSP. By whole-exome sequencing, we sequencing identified a nonsense variation c.412C > T (p.Arg138*) in the phosphomevalonate kinase gene (PMVK), which encodes a cytoplasmic enzyme catalyzing the conversion of mevalonate 5-phosphate to mevalonate 5-diphosphate in the mevalonate pathway. By co-segregation and haplotype analyses as well as exclusion testing of 500 normal control subjects, we demonstrated that this genetic variant was involved in the development of DSP in both families. We obtained further evidence from studies using HaCaT cells as models that this variant disturbed subcellular localization, expression and solubility of PMVK. We also observed apparent apoptosis in and under the cornoid lamella of PMVK-deficient lesional tissues, with incomplete differentiation of keratinocytes. Our findings suggest that PMVK is a potential novel gene involved in the pathogenesis of DSP and PMVK deficiency or abnormal keratinocyte apoptosis could lead to porokeratosis.

Mutations of Ceramide kinase-like ( CERKL ) gene are associated with retinitis pigmentosa (RP), an inherited degenerative eye disease. CERKL encodes an antioxidant protein which is critical to photoreceptor survival, its deficiency causes retinal degeneration as a result of oxidative damage. However, the regulation of CERKL in response to oxidative stress, and its contribution to photoreceptor survival remain unclear. pVHL, the substrate receptor of RING finger-type SCF like ECV ubiquitin ligase, binds and ubiquitinates a number of hydroxylated proteins for proteasomal degradation. Due to hydroxylated proteins which are modified by PHD1–3, pVHL dependent ubiquitin-proteasomal degradation pathway is blocked by PHD1-3 inhibitors (e.g. hypoxia or oxidative stress). In this study, we identified pVHL as an important regulator of CERKL. Western blot and in vivo ubiquitination assays showed hypoxia up-regulates CERKL at protein level by down-regulating its poly-ubiquitination. By Co-IP and domain mapping studies, we found CERKL complexes with ECV ligase via pVHL. Through overexpression and small RNA interference analysis, we demonstrated pVHL ubiquitinates CERKL for proteasomal degradation. Additionally, our work showed that the oxygen sensors PHD1 and PHD3 are involved in CERKL degradation. Collectively, our results indicated that pVHL interacts with CERKL and ubiquitinates it for oxygen dependent proteasomal degradation.