Pinoresinol diglucoside mitigates dexamethasone-induced osteoporosis and chondrodysplasia in zebrafish.

BACKGROUND: The global increase in the aging population has led to a higher incidence of osteoporosis among the elderly. OBJECTIVE: This study aimed to evaluate the protective properties of pinoresinol diglucoside (PDG), an active constituent of Eucommia ulmoides, against dexamethasone-induced osteoporosis and chondrodysplasia. METHODS: A zebrafish model of osteoporosis was established by exposing larval zebrafish to dexamethasone. The impact of PDG on bone mineralization was assessed through alizarin red and calcein staining. Alkaline phosphatase activity was quantified to evaluate osteoblast function. The influence of PDG on chondrogenesis was estimated using alcian blue staining. Fluorescence imaging and motor behavior analysis were employed to assess the protective effect of PDG on the structure and function of dexamethasone-induced skeletal teratogenesis. qPCR determined the expression of osteogenesis and Wnt signaling-related genes. Molecular docking was used to assess the potential interactions between PDG and Wnt receptors. RESULTS: PDG significantly increased bone mineralization and corrected spinal curvature and cartilage malformations in the zebrafish model. Furthermore, PDG enhanced swimming abilities compared to the model group. PDG mitigated dexamethasone-induced skeletal abnormalities in zebrafish by upregulating Wnt signaling, showing potential interaction with Wnt receptors FZD2 and FZD5. CONCLUSION: PDG mitigates dexamethasone-induced osteoporosis and chondrodysplasia by promoting bone formation and activating Wnt signaling.

Hexafluoropropylene oxide trimer acid, a perfluorooctanoic acid alternative, induces cardiovascular toxicity in zebrafish embryos.

As an increasingly used alternative to perfluorooctanoic acid (PFOA), hexafluoropropylene oxide trimer acid (HFPO-TA) has been widely detected in global water environments. However, little is known regarding its toxic effects on cardiovascular development. Here, zebrafish embryos were treated with egg water containing 0, 60, 120, or 240 mg/L HFPO-TA. Results showed that HFPO-TA treatment led to a significant reduction in both larval survival percentage and heart rate. Furthermore, HFPO-TA exposure caused severe pericardial edema and elongation of the sinus venous to bulbus arteriosus distance (SV-BA) in Tg (myl7: GFP) transgenic larvae, disrupting the expression of genes involved in heart development and thus causing abnormal heart looping. Obvious sprouting angiogenesis was observed in the 120 and 240 mg/L exposed Tg (fli: GFP) transgenic larvae. HFPO-TA treatment also impacted the mRNA levels of genes involved in the vascular endothelial growth factor (VEGF) pathway and embryonic vascular development. HFPO-TA exposure significantly decreased erythrocyte number in Tg (gata1: DsRed) transgenic embryos and influenced gene expression associated with the heme metabolism pathway. HFPO-TA also induced oxidative stress and altered the transcriptional levels of genes related to cell cycle and apoptosis, inhibiting cell proliferation while promoting apoptosis. Therefore, HFPO-TA exposure may induce abnormal development of the cardiovascular and hematopoietic systems in zebrafish embryos, suggesting it may not be a suitable or safe alternative for PFOA.

Sanguinarine exposure induces immunotoxicity and abnormal locomotor behavior in zebrafish.

Sanguinarine (C20H14NO4+), a plant alkaloid and pesticide, works well a fungicidal and insecticidal applications. The prospect that sanguinarine may have potentially toxic effects on aquatic organisms has been brought to light by its use in agriculture. The first evaluation of the immunotoxic and behavioral effects of sanguinarine exposure on larval zebrafish was done in this work. Firstly, zebrafish embryos exposed to sanguinarine had shorter body length, larger yolk sacs, and slower heart rates. Secondly, the number of innate immune cells was significantly reduced. Thirdly, alterations in locomotor behavior were observed as exposure concentrations increased. Total distance travelled, travel time, and mean speed were all reduced. We also found significant changes in oxidative stress-related indicators and a significant increase in apoptosis in the embryos. Further studies revealed aberrant expression of some key genes in the TLR immune signaling pathway including CXCL-c1c, IL8, MYD88, and TLR4. At the same time, the expression of the pro-inflammatory cytokine IFN-γ was upregulated. To sum up, our results suggest that sanguinarine exposure may cause immunotoxicity and aberrant behavior in larval zebrafish.

FYCO1 regulates migration, invasion, and invadopodia formation in HeLa cells through CDC42/N-WASP/Arp2/3 signaling pathway.

FYCO1, an autophagy adaptor, plays an essential role in the trafficking toward the plus-end of microtubules and the fusion of autophagosomes. Autophagic dysfunction is involved in numerous disease states, including cancers. Previous studies have implicated FYCO1 as one of the critical genes involved in the adenoma to carcinoma transition, but the biological function and mechanism of FYCO1 in carcinogenesis remain unclear. This study aims to elucidate the role and mechanism of up- and downregulation of FYCO1 in mediating tumor effects in HeLa cells. Functionally, FYCO1 promotes cellular migration, invasion, epithelial-mesenchymal transition, invadopodia formation, and matrix degradation, which are detected through wound healing, transwell, immunofluorescence, and Western blot approaches. Interestingly, the data show that although FYCO1 does not affect HeLa cell proliferation, cell cycle distribution, nor vessels' formation, FYCO1 can block the apoptotic function. FYCO1 inhibits cleavage of PARP, caspase3, and caspase9 and increases Bcl-2/Bax ratio. Then, we used CK666, an Arp2/3 specific inhibitor, to confirm that FYCO1 may promote the migration and invasion of HeLa cells through the CDC42/N-WASP/Arp2/3 signaling pathway. Taken together, these results provide a new insight that FYCO1, an autophagy adaptor, may also be a new regulator of tumor metastasis.

Identification and functional analysis of two GJA8 variants in Chinese families with eye anomalies.

In this study, we report on two different GJA8 variants related to congenital eye anomalies in two unrelated families, respectively. GJA8 (or Cx50) encoding a transmembrane protein to form lens connexons has been known as a common causative gene in congenital cataracts and its variants have recently been reported related to a wide phenotypic spectrum of eye defects. We identified two GJA8 variants, c.134G>T (p.Try45Leu, W45L) detected in a cataract family by Sanger sequencing and c.281G>A (p.Gly94Glu, G94E) found in a family with severe eye malformations including microphthalmia by whole-exome sequencing. These two variants were absent in healthy population and predicted deleterious by bioinformatic analysis. Furthermore, we compared the expression in cell lines between these mutants and the wildtype to explore their potential mechanism. Cell counting kit-8 assay showed that overexpression of either W45L or G94E decreased cell viability compared with wild-type Cx50 and the control. A lower protein level in W45L found by western blotting and fewer punctate fluorescent signals showed by fluorescence microscopy suggested that W45L may have less protein expression. A higher G94E protein level and abundant dotted distribution indicated that G94E may cause aberrant protein degradation and accumulation. Such results from in vitro assays confirmed the impact of these two variants and gave us a hint about their different pathogenic roles in different phenotypes. In conclusion, our study is the first to have the functional analysis of two GJA8 variants c.134G>T and c.281G>A in Chinese pedigrees and explore the impact of these variants, which can help in prenatal diagnosis and genetic counseling as well in basic studies on GJA8.

Identification and functional study of FOXC1 variants in Chinese families with glaucoma.

This study aimed to identify the disease-causing gene of three Chinese families with glaucoma. Whole exome sequencing was performed on the probands and detected three different variants (c.405C>A (p.Cys135Ter), c.851G>T (p.Ser284Ile), and c.392C>T (p.Ser131Leu)) in FOXC1 as a causative gene of glaucoma, and Sanger sequencing was performed for verification and cosegregation analysis. Three in silico tools all predicted these two missense variants to be probably disease-causing. Western blot analysis, immunofluorescence, and dual-luciferase assay were further used to evaluate the effect of FOXC1 missense variants, and demonstrated that the two variants resulted in decreased transactivation activity of FOXC1 although the variants had no effect on the protein amount and the nucleus subcellar localization of FOXC1 compared with the wild type, which implies that both of two variants may be probably pathogenic. In this study, we reported two novel FOXC1 variants as well as a reported variant and the phenotypes associated to these variants, which expands the spectrum and relevant phenotypes of FOXC1 variants. Additionally, the functional analysis of FOXC1 variants provides further insight into the possible pathogenesis of anterior segment anomaly related to FOXC1.

Exposure to pyrazosulfuron-ethyl induces immunotoxicity and behavioral abnormalities in zebrafish embryos.

Pyrazosulfuron-ethyl is one of the most widely used herbicides in agriculture and can be widely detected in aquatic ecosystems. However, its biosafety, including its potential toxic effects on aquatic organisms and its mechanism, is still poorly understood. As an ideal vertebrate model, zebrafish, the effect of pyrazosulfuron-ethyl on early embryonic development and immunotoxicity of zebrafish can be well evaluated. From 10 to 72 h post fertilization (hpf), zebrafish embryos were exposed to 1, 5, and 9 mg/L pyrazosulfuron-ethyl which led in a substantial reduction in survival, total length, and heart rate, as well as a range of behavioral impairments. In zebrafish larvae, the number of neutrophils and macrophages was considerably decreased and oxidative stress levels increased in a dose-dependent way after pyrazosulfuron-ethyl exposure. And the expression of immune-related genes, such as TLR-4, MyD88 and IL-1ß, were downregulated by pyrazosulfuron-ethyl exposure. Moreover, pyrazosulfuron-ethyl exposure also inhibited motor behavior. Notch signaling was upregulated after exposure to pyrazosulfuron-ethyl, while inhibition of Notch signaling pathway could rescue immunotoxicity. Therefore, our findings suggest that pyrazosulfuron-ethyl has the potential to induce immunotoxicity and neurobehavioral changes in zebrafish larvae.

Mutation screening of the USH2A gene reveals two novel pathogenic variants in Chinese patients causing simplex usher syndrome 2.

BACKGROUND: Usher syndrome (USH) is the most prevalent cause of the human genetic deafness and blindness. USH type II (USH2) is the most common form of USH, and USH2A is the major pathogenic gene for USH2. For expanding the spectrum of USH2A mutations and further revealing the role of USH2A in USH2, we performed the USH2A gene variant screening in Chinese patients with USH2. METHODS: Genomic DNA was extracted from peripheral blood of unrelated Chinese USH2 patients, we designed specific primers for amplifying the coding region (exons 2-72) of the USH2A gene. Sanger sequencing was used to study alleles. Silico prediction tools were used to predict the pathogenicity of the variants identified in these patients. RESULTS: Five heterozygous pathogenic variants were detected in four patients. Two patients were found to have two-mutations and two patients only have one. Two novel variants c.4217C > A (p.Ser1406X) and c.11780A > G (p.Asp3927Gly)) were predicted deleterious by computer prediction algorithms. In addition, three reported mutations (c.8559-2A > G, c.8232G > C and c.11389 + 3A > T) were also found in this study. CONCLUSIONS: We identified five heterozygous pathogenic variants in the USH2A gene in Chinese patients diagnosed with Usher syndrome type 2, two of which were not reported. It expands the spectrum of USH2A variants in USH.

Seven novel variants expand the spectrum of RPE65-related Leber congenital amaurosis in the Chinese population.

Purpose: To screen RPE65 in 187 families with Leber congenital amaurosis (LCA). Methods: Sanger sequencing and/or targeted exome sequencing was employed to identify mutations in the RPE65 gene, and intrafamilial cosegregation analysis if DNA was available. In silico analyses and splicing assay were used to evaluate the variants' pathogenicity. Results: Genetic analysis revealed 15 mutations in RPE65 in 14 pedigrees, including one splice-site mutation, one frameshift mutation, three nonsense mutations, and ten missense mutations. Of the mutations identified in RPE65, seven are novel associated with LCA, including five missense variants (c.124C>T, c.149T>C, c.340A>C, c.425A>G, and c.1399C>G) and two indel (insertions or deletions) variants (c.858+1delG and c.1181_1182insT). In vitro splicing assay was performed to evaluate the functional impact on RNA splicing of novel mutations if two of three in silico analyses were predicated to be non-pathogenic at the protein level. Among these 15 variants, 14 were classified as 'pathogenic variants,' and a variant (c.124C>T) was 'variants with uncertain significance' according to the standards and guidelines of the American College of Medical Genetics and Genomics. Conclusions: Mutations in RPE65 were responsible for 11 of the cohort of 187 Chinese families with LCA, which expands the spectrum of RPE65-related LCA in the Chinese population and potentially facilitates its clinical implementation.

Identification of a novel PAX6 mutation in a Chinese family with aniridia.

BACKGROUND: This study aims to investigate the clinical characterization and causative genetic defect of a four-generation Chinese family with autosomal dominant aniridia. METHODS: The recruited family members underwent comprehensive routine and ophthalmic examinations, and Sanger sequencing was performed to screen the mutation in PAX6. RESULTS: A novel heterozygous PAX6 deletion c.435_445delTAGCGAAAAGC (p.Ser146ThrfsX9) in exon 7 was identified in all affected individuals, but this was absent in any of the unaffected family members and in the 200 unrelated controls. CONCLUSION: A novel deletion in the PAX6 gene was identified in a Chinese family associated with aniridia, which expands the spectrum of the PAX6 mutation and its associated phenotype.

Benzophenone induces cardiac developmental toxicity in zebrafish embryos by upregulating Wnt signaling.

Benzophenone (BP) is found in many popular consumer products, such as cosmetics. BP potential toxicity to humans and aquatic organisms has emerged as an increased concern. In current study, we utilized a zebrafish model to assess BP-induced developmental cardiotoxicity. Following BP exposure, zebrafish embryos exhibited developmental toxicity, including increased mortality, reduced hatchability, delayed yolk sac absorption, and shortened body length. Besides, BP exposure induced cardiac defects in zebrafish embryos, comprising pericardial edema, reduced myocardial contractility and rhythm disturbances, and altered expression levels of cardiac developmental marker genes. Mechanistically, BP exposure disturbed the redox state and increased the level of apoptosis in zebrafish cardiomyocytes. Transcriptional expression levels of Wnt signaling genes, involving lef1, axin2, and ß-catenin, were upregulated after BP treatment. Inhibition of Wnt signaling with IWR-1 could rescue the BP-induced cardiotoxicity in zebrafish. In summary, BP exposure causes cardiotoxicity via upregulation of the Wnt signaling pathway in zebrafish embryos.

Hexafluoropropylene oxide trimer acid (HFPO-TA) disturbs embryonic liver and biliary system development in zebrafish.

Hexafluoropropylene oxide trimer acid (HFPO-TA), a novel alternative to perfluorooctanoic acid (PFOA), has emerged as a potential environmental pollutant. Here, to investigate the toxic effects of HFPO-TA on liver and biliary system development, zebrafish embryos were exposed to 0, 50, 100, or 200 mg/L HFPO-TA from 6 to 120 h post-fertilization (hpf). Results showed that the 50 % lethal concentration (LC50) of HFPO-TA was 231 mg/L at 120 hpf, lower than that of PFOA. HFPO-TA exposure decreased embryonic hatching, survival, and body length. Furthermore, HFPO-TA exerted higher toxicity at the specification stage than during the differentiation and maturation stages, leading to small-sized livers in Tg(fabp10a: DsRed) transgenic larvae and histopathological changes. Significant decreases in the mRNA expression of genes related to liver formation were observed. Alanine transaminase (ALT), aspartate aminotransferase (AST), total bilirubin (TBIL), and direct bilirubin (DBIL) levels were significantly increased. HFPO-TA decreased total cholesterol (TCHO) and triglyceride (TG) activities, disturbed lipid metabolism through the peroxisome proliferator-activated receptor (PPAR) pathway, and induced an inflammatory response. Furthermore, HFPO-TA inhibited intrahepatic biliary development in Tg(Tp1:eGFP) transgenic larvae and interfered with transcription of genes associated with biliary duct development. HFPO-TA reduced bile acid synthesis but increased bile acid transport, resulting in disruption of bile acid metabolism. Therefore, HFPO-TA influenced embryonic liver and biliary system morphogenesis, caused liver injury, and may be an unsafe alternative for PFOA.

Hepatic lipid metabolism disorders and immunotoxicity induced by cysteamine in early developmental stages of zebrafish.

Cysteamine, a sulfhydryl compound, is an intermediate in the metabolism of coenzyme A to taurine in living organisms. However, the potential side effects of cysteamine such as hepatotoxicity in pediatric patients have been reported in some studies. To evaluate the impact of cysteamine on infants and children, larval zebrafish (a vertebrate model) were exposed to 0.18, 0.36 and 0.54 mM cysteamine from 72 hpf to 144 hpf. Alterations in general and pathological evaluation, biochemical parameters, cell proliferation, lipid metabolism factors, inflammatory factors and Wnt signaling pathway levels were examined. Increased liver area and lipid accumulation were observed in liver morphology, staining and histopathology in a dose-dependent manner with cysteamine exposure. In addition, the experimental cysteamine group exhibited higher alanine aminotransferase, aspartate aminotransferase, total triglyceride and total cholesterol levels than the control group. Meanwhile, the levels of lipogenesis-related factors ascended whereas lipid transport-related factors descended. Oxidative stress indicators such as reactive oxygen species, MDA and SOD were upregulated after cysteamine exposure. Afterwards, transcription assays revealed that biotinidase and Wnt pathway-related genes were upregulated in the exposed group, and inhibition of Wnt signaling partially rescued the abnormal liver development. The current study found that cysteamine-induced hepatotoxicity in larval zebrafish is due to inflammation and abnormal lipid metabolism, which is mediated by biotinidase (a potential pantetheinase isoenzyme) and Wnt signaling. This provides a perspective on the safety of cysteamine administration in children and identifies potential targets for protection against adverse reactions.

Cysteamine hydrochloride affects ocular development and triggers associated inflammation in zebrafish.

The increasing use of cosmetics has raised widespread concerns regarding their ingredients. Cysteamine hydrochloride (CSH) is a newly identified allergenic component in cosmetics, and therefore its potential toxicity needs further elucidation. Here, we investigated the in vivo toxicity of CSH during ocular development utilizing a zebrafish model. CSH exposure was linked to smaller eyes, increased vasculature of the fundus and decreased vessel diameter in zebrafish larvae. Moreover, CSH exposure accelerated the process of vascular sprouting and enhanced the proliferation of ocular vascular endothelial cells. Diminished behavior in response to visual stimuli and ocular structural damage in zebrafish larvae after CSH treatment were confirmed by analysis of the photo-visual motor response and pathological examination, respectively. Through transcriptional assays, transgenic fluorescence photography and molecular docking analysis, we determined that CSH inhibited Notch receptor transcription, leading to an aberrant proliferation of ocular vascular endothelial cells mediated by Vegf signaling activation. This process disrupted ocular homeostasis, and induced an inflammatory response with neutrophil accumulation, in addition to the generation of high levels of reactive oxygen species, which in turn promoted the occurrence of apoptotic cells in the eye and ultimately impaired ocular structure and visual function during zebrafish development.

Identification of 12 OCA Cases in Chinese Population and Two Novel Variants.

OCA (oculocutaneous albinism) refers to a group of heterogeneous congenital disorders of which the common manifestations are variable degrees of cutaneous hypopigmentation and significant visual impairment, including poor visual acuity, photophobia, and nystagmus. Molecular analysis may elucidate its pathogenesis and be in favor of accurate diagnosis. High-throughput sequencing and Sanger sequencing were performed to detect mutational alleles and in silico analysis was performed for prediction of variant pathogenicity. Ten TYR-related and two OCA2-related patients were identified with 16 different variants with potential pathogenicity. Two novel missense variants [TYR: c.623T > G, p(Leu208Arg) and OCA2: c.1325A > G, p(Asn442Ser)] are identified in this study, and three OCA cases are reported for the first time in Chinese population based on their associated variants. Analysis of crystal structures of TYR ortholog and its paralog TYRP1 suggests that the substitution of Leu208 may have an impact on protein stability. This study may facilitate OCA diagnosis by expanding the mutational spectrum of TYR and OCA2 as well as further basic studies about these two genes.

Propranolol hydrochloride induces neurodevelopmental toxicity and locomotor disorders in zebrafish larvae.

Propranolol hydrochloride is the first-line drug for the clinical treatment of hypertension, arrhythmia, and other diseases. However, with the increasing use of this drug, its safety and environmental health have received more and more attention. In this study, aquatic vertebrate zebrafish were used as a model to study the toxic effects and mechanisms of propranolol hydrochloride. It was revealed that zebrafish larvae exposed to propranolol hydrochloride showed aberrant head nerve development and locomotor disorders. Additionally, exposure to propranolol hydrochloride could induce oxidative stress, alter the activities of AChE and ATPase, and disrupt the expression of genes involved in neurodevelopment and neurotransmitter pathways. More interestingly, the expression of Parkinson's disease-related genes was altered in zebrafish treated with propranolol hydrochloride. We detected the expression of genes related to the Wnt signaling pathway and found that their expression appeared to be down-regulated. The phenotype of nerve developmental defects and locomotor disorders can be effectively rescued by astaxanthin and Wnt activators. Collectively, the results suggest that propranolol hydrochloride may induce neurotoxicity and abnormal movement behavior with PD-like symptoms in zebrafish larvae.

Cysteamine affects skeletal development and impairs motor behavior in zebrafish.

Cysteamine is a kind of feed additive commonly used in agricultural production. It is also the only targeted agent for the treatment of cystinosis, and there are some side effects in clinical applications. However, the potential skeletal toxicity remains to be further elucidated. In this study, a zebrafish model was for the first time utilized to synthetically appraise the skeletal developmental defects induced by cysteamine. The embryos were treated with 0.35, 0.70, and 1.05 mM cysteamine from 6 h post fertilization (hpf) to 72 hpf. Substantial skeletal alterations were manifested as shortened body length, chondropenia, and abnormal somite development. The results of spontaneous tail coiling at 24 hpf and locomotion at 120 hpf revealed that cysteamine decreased behavioral abilities. Moreover, the level of oxidative stress in the skeleton ascended after cysteamine exposure. Transcriptional examination showed that cysteamine upregulated the expression of osteoclast-related genes but did not affect osteoblast-related genes expression. Additionally, cysteamine exposure caused the downregulation of the Notch signaling and activating of Notch signaling partially attenuated skeletal defects. Collectively, our study suggests that cysteamine leads to skeletal developmental defects and reduces locomotion activity. This hazard may be associated with cysteamine-mediated inhibition of the Notch signaling and disorganization of notochordal cells due to oxidative stress and apoptosis.

A novel BLOC1S5-related HPS-11 patient and zebrafish with bloc1s5 disruption.

Hermansky-Pudlak Syndrome (HPS) cases present with a variable degree of OCA and bleeding tendency. HPS is categorized into eleven types based on eleven causative genes, and disease severity varies among different types. By whole-exome sequencing performed on a family trio and Sanger sequencing of candidate variants, we identified a novel homozygous variant (NM_201280.3: c.181delC, p.Val61*) in BLOC1S5 in the patient who presents OCA and mild bleeding diathesis, and his healthy parents are heterozygous carriers. The variant can be considered pathogenic based on the guideline American College of Medical Genetics and Genomics, and the patient is proposed to be affected with HPS-11. In this study, we also explored bloc1s5 in zebrafish. bloc1s5 mRNA can be detected during early development of zebrafish. bloc1s5 knockdown zebrafish present with retinal hypopigmentation, thrombocytes loss and pericardial edema, and dll4/notch1 signaling and vascular integrity signaling are down-regulated at mRNA level in bloc1s5 morphants. The data from the first HPS-11 patient in Chinese population expand phenotypic and genotypic spectrum of HPS-11. Disruption of bloc1s5 in zebrafish recapitulates HPS-11-like phenotypes, and the potential signaling pathways associated with bloc1s5 are proposed. Altogether, this study may facilitate genetic counseling of HPS and investigation about BLOC1S5.

Comprehensive analysis of spectral distribution of a large cohort of Chinese patients with non-syndromic oculocutaneous albinism facilitates genetic diagnosis.

Non-syndromic oculocutaneous albinism (nsOCA) is a group of genetically heterogeneous autosomal recessive disorders with complete lack or decrease pigmentation in skin, hair, and eyes. TYR, OCA2, TYRP1, SLC45A2, SLC24A5, and LRMDA were reported to cause OCA1-4 and OCA6-7, respectively. By sequencing all the known nsOCA genes in 114 unrelated Chinese nsOCA patients combined with In silico analyses, splicing assay, and classification of variants according to the standards and guidelines of American College of Medical Genetics and Genomics, we detected seventy-one different OCA-causing variants separately in TYR, OCA2, SLC45A2, and SLC24A5, including thirty-one novel variants (13 in TYR, 11 in OCA2, and 7 in SLC45A2). This study shows that OCA1 is the most common (75/114) and OCA2 ranks the second most common (16/114) in Chinese. 99 patients of our cohort were caused by variants of all the known nsOCA genes. Cutaneous phenotypes of OCA1, OCA2, and OCA4 patients were shown in this study. The second OCA6 case in China was identified here. These data expand the spectrum of OCA variants as well phenotype and facilitate clinical implement of Chinese OCA patients.

PITX3 mutations associated with autosomal dominant congenital cataract in the Chinese population.

The present study aimed to identify the disease­causing gene of a four­generation Chinese family affected with congenital posterior subcapsular cataracts (CPSC), to additionally investigate the frequency of paired like homeodomain 3 (PITX3) mutations in Chinese patients with autosomal dominant congenital cataract (ADCC) and to analyze the pathogenesis of the mutations identified in the present study. Whole exome sequencing (WES) was utilized to identify the genetic cause of CPSC in the four­generation family. Sanger sequencing was performed to verify the WES results and to screen for mutations of the PITX3 gene in probands of an additional 194 Chinese ADCC families. Co­segregation analysis was performed in the family members with available DNA. Subcellular localization analyses and transactivation assays were performed for the PITX3 mutations identified. From the WES data, the c.608delC (p.A203GfsX106) mutation of PITX3 was identified in the four­generation family with CPSC. A second PITX3 mutation c.640_656del (p.A214RfsX42) was detected in two of the additional 194 ADCC families and one of these two families exhibited incomplete penetrance. Functional studies indicated that these 2 PITX3 mutant proteins retained a nuclear localization pattern, but resulted in decreased transactivation activity, similar to other previously identified PITX3 mutations. In the present study, 2 different mutations (p.A203GfsX106 and p.A214RfsX42) in PITX3 were identified as the causative defect in a four­generation family with CPSC and two ADCC families, respectively. The prevalence of PITX3 gene­associated cataract was 1.54% (3/195) in the Chinese congenital cataract (CC) family cohort. In vitro functional analyses of these 2 PITX3 mutations were performed, in order to enhance understanding of the pathogenesis of CC caused by PITX3 mutations.