Prognostic and diagnostic value of circulating IGFBP2 in pancreatic cancer.

Insulin-like growth factor binding protein 2 (IGFBP2) is overexpressed in tumor tissues of several malignancies, including pancreatic cancer. Because of its role in tumor progression, IGFBP2 has been investigated as a tumor biomarker. However, little is known about its utility in pancreatic cancer. Plasma IGFBP2 levels were determined using enzyme-linked immunosorbent assay in 75 patients with pancreatic ductal adenocarcinoma (PDAC), 73 matched healthy controls, and 17 chronic pancreatitis patients. Our results showed that the plasma IGFPB2 level was significantly higher in PDAC patients than in patients with chronic pancreatitis and healthy controls. At a cut-off value of 333.9 ng/mL, the specificity and sensitivity were 78.08 and 65.33%, respectively. IGFBP2 level alone did not outperform carbohydrate antigen 19-9 (CA19-9) in diagnostic accuracy, but it successfully identified 9 out of 24 PDAC patients who were misidentified by CA19-9. The combination of IGFBP2 and CA19-9 was more accurate in the detection of PDAC than CA19-9 alone. IGFBP2 was more accurate than the other in discriminating between chronic pancreatitis and PDAC. Plasma IGFBP2, rather than CA19-9, was higher in the new-onset diabetes, lymph node involvement, and distant metastasis subgroups. IGFBP2 level was notably higher in stage IV cases than in stage I/II or stage III disease. However, CA19-9 did not show a difference between stages. After adjusting for lymph node involvement and distant metastasis, plasma IGFBP2 was identified as an independent prognostic marker for PDAC. The median survival time for patients with an IGFBP2 level ≥333.9 ng/mL was significantly shorter than that for patients with an IGFBP2 level <333.9 ng/mL. Marked elevation of plasma IGFBP2 in PDAC is associated with poorer survival. IGFBP2 may be considered as a supplementary biomarker for the diagnosis and prognostic prediction in Chinese pancreatic cancer patients.

The RhoB p.S73F mutation leads to cerebral palsy through dysregulation of lipid homeostasis.

Cerebral palsy (CP) is a prevalent neurological disorder that imposes a significant burden on children, families, and society worldwide. Recently, the RhoB p.S73F mutation was identified as a de novo mutation associated with CP. However, the mechanism by which the RhoB p.S73F mutation causes CP is currently unclear. In this study, rabbit models were generated to mimic the human RhoB p.S73F mutation using the SpG-BE4max system, and exhibited the typical symptoms of human CP, such as periventricular leukomalacia and spastic-dystonic diplegia. Further investigation revealed that the RhoB p.S73F mutation could activate ACAT1 through the LYN pathway, and the subsequently altered lipid levels may lead to neuronal and white matter damage resulting in the development of CP. This study presented the first mammalian model of genetic CP that accurately replicates the RhoB p.S73F mutation in humans, provided further insights between RhoB and lipid metabolism, and novel therapeutic targets for human CP.

Clinical outcomes of second-line chemotherapy in patients with advanced pancreatic adenocarcinoma: a real-world study.

OBJECTIVE: Little progress has been made in recent years using first-line chemotherapy, including gemcitabine combined with nab-paclitaxel, FOLFIRINOX, and NALIRIFOX, for advanced pancreatic adenocarcinoma (APC). In addition, the optimal second-line chemotherapy regimen has not been determined. This study aimed to compare the effectiveness of different types of second-line chemotherapy for APC. METHODS: Patients with APC who received first-line treatment from January 2008 to January 2021 were considered eligible for this retrospective analysis. The primary and secondary endpoints were overall survival (OS) and progression-free survival (PFS), respectively. RESULTS: Four hundred and thirty-seven and 617 patients were treated with 5-fluorouracil- and gemcitabine-based chemotherapy as first-line treatment, respectively. Demographic and clinical features, except age and liver metastasis, were comparable between the two groups (P < 0.05). The median OS was 8.8 and 7.8 months in patients who received a 5-fluorouracil- and gemcitabine-based combined regimen for first-line therapy, respectively (HR = 1.244, 95% CI = 1.090-1.419; P < 0.001). The median OS was 5.6 and 1.9 months in patients who received second-line chemotherapy and supportive care, respectively (HR = 0.766, 95% CI = 0.677-0.867; P < 0.001). The median PFS was not significantly differently between gemcitabine or 5-fluorouracil monotherapy and combination therapy. CONCLUSIONS: A 5-fluorouracil- or gemcitabine-based combined regimen was shown to be as effective as a single 5-fluorouracil or gemcitabine regimen as second-line therapy for patients with APC.

Gene editing therapy for cardiovascular diseases.

The development of gene editing tools has been a significant area of research in the life sciences for nearly 30 years. These tools have been widely utilized in disease detection and mechanism research. In the new century, they have shown potential in addressing various scientific challenges and saving lives through gene editing therapies, particularly in combating cardiovascular disease (CVD). The rapid advancement of gene editing therapies has provided optimism for CVD patients. The progress of gene editing therapy for CVDs is a comprehensive reflection of the practical implementation of gene editing technology in both clinical and basic research settings, as well as the steady advancement of research and treatment of CVDs. This article provides an overview of the commonly utilized DNA-targeted gene editing tools developed thus far, with a specific focus on the application of these tools, particularly the clustered regularly interspaced short palindromic repeat/CRISPR-associated genes (Cas) (CRISPR/Cas) system, in CVD gene editing therapy. It also delves into the challenges and limitations of current gene editing therapies, while summarizing ongoing research and clinical trials related to CVD. The aim is to facilitate further exploration by relevant researchers by summarizing the successful applications of gene editing tools in the field of CVD.

Noninvasive Light Flicker Stimulation Promotes Optic Nerve Regeneration by Activating Microglia and Enhancing Neural Plasticity in Zebrafish.

Purpose: Forty-hertz light flicker stimulation has been proven to reduce neurodegeneration, but its effect on optic nerve regeneration is unclear. This study explores the effect of 40-Hz light flicker in promoting optic nerve regeneration in zebrafish and investigates the underlying mechanisms. Methods: Wild-type and mpeg1:EGFP zebrafish were used to establish a model of optic nerve crush. Biocytin tracing and hematoxylin and eosin staining were employed to observe whether 40-Hz light flicker promotes regeneration of retinal ganglion cell axons and dendrites. Optomotor and optokinetic responses were evaluated to assess recovery of visual function. Immunofluorescence staining of mpeg1:EGFP zebrafish was performed to observe changes in microglia. Differentially expressed genes that promote optic nerve regeneration following 40-Hz light flicker stimulation were identified and validated through RNA-sequencing analysis and quantitative real-time PCR (qRT-PCR). Results: Zebrafish exhibited spontaneous optic nerve regeneration after optic nerve injury and restored visual function. We observed that 40-Hz light flicker significantly activated microglia following optic nerve injury and promoted regeneration of retinal ganglion cell axons and dendrites, as well as recovery of visual function. Transcriptomics and qRT-PCR analyses revealed that 40-Hz light flicker increased the expression of genes associated with neuronal plasticity, including bdnf, npas4a, fosab, fosb, egr4, and ier2a. Conclusions: To our knowledge, this study is the first to demonstrate that 40-Hz light flicker stimulation promotes regeneration of retinal ganglion cell axons and dendrites and recovery of visual function in zebrafish, which is associated with microglial activation and enhancement of neural plasticity.

EGR1 and EGR2 positively regulate plant ABA signaling by modulating the phosphorylation of SnRK2.2.

During abscisic acid (ABA) signaling, reversible phosphorylation controls the activity and accumulation of class III SNF1-RELATED PROTEIN KINASE 2s (SnRK2s). While protein phosphatases that negatively regulate SnRK2s have been identified, those that positively regulate ABA signaling through SnRK2s are less understood. In this study, Arabidopsis thaliana mutants of Clade E Growth-Regulating 1 and 2 (EGR1/2), which belong to the protein phosphatase 2C family, exhibited reduced ABA sensitivity in terms of seed germination, cotyledon greening, and ABI5 accumulation. Conversely, overexpression increased these ABA-induced responses. Transcriptomic data revealed that most ABA-regulated genes in egr1 egr2 plants were expressed at reduced levels compared with those in Col-0 after ABA treatment. Abscisic acid up-regulated EGR1/2, which interact directly with SnRK2.2 through its C-terminal domain I. Genetic analysis demonstrated that EGR1/2 function through SnRK2.2 during ABA response. Furthermore, SnRK2.2 de-phosphorylation by EGR1/2 was identified at serine 31 within the ATP-binding pocket. A phospho-mimic mutation confirmed that phosphorylation at serine 31 inhibited SnRK2.2 activity and reduced ABA responsiveness in plants. Our findings highlight the positive role of EGR1/2 in regulating ABA signaling, they reveal a new mechanism for modulating SnRK2.2 activity, and provide novel insight into how plants fine-tune their responses to ABA.

Protective effect of the natural flavonoid naringenin in mouse models of retinal injury.

Glaucoma is an eye disease with a high rate of blindness and a complex pathogenesis. Ocular hypertension (OHT) is a critical risk factor, and retinal ischemia/reperfusion (I/R) is an important pathophysiological basis. This study was designed to investigate the retinal neuroprotective effect of oral naringenin in an acute retinal I/R model and a chronic OHT model and the possible mechanism involved. After the I/R and OHT models were established, mice were given vehicle or naringenin (100 mg/kg or 300 mg/kg). Hematoxylin-eosin (HE) staining and immunostaining of RBPMS and glial fibrillary acidic protein (GFAP) were used to evaluate retinal injury. GFAP, CD38, Sirtuin1 (SIRT1), and NOD-like receptor protein 3 (NLRP3) expression levels were measured by Western blotting. In the OHT model, intraocular pressure (IOP) was dynamically maintained at approximately 20-25 mmHg after injury. The retinal structure was damaged, and retinal ganglion cells (RGCs) were lost in both models. Naringenin ameliorated the abovementioned indications but also demonstrated that high concentrations of naringenin significantly inhibited retinal astrocyte activation and inhibited damage-induced increases in the expression of GFAP, NLRP3, and CD38 proteins, while SIRT1 protein expression was upregulated. This study showed for the first time that naringenin can reduce microbead-induced IOP elevation in the OHT model, providing new evidence for the application of naringenin in glaucoma. Naringenin may mediate the CD38/SIRT1 signaling pathway, inhibit astrocyte activation, and ultimately exert an anti-inflammatory effect to achieve retinal neuroprotection.

Resveratrol Alleviates Retinal Ischemia-Reperfusion Injury by Inhibiting the NLRP3/Gasdermin D/Caspase-1/Interleukin-1ß Pyroptosis Pathway.

Purpose: The purpose of this study is to investigate the anti-pyroptotic effect of resveratrol in the context of ischemia-reperfusion (I/R)-induced retinal injury, with a particular focus on Müller glial cells (MGCs) and to elucidate the underlying molecular mechanisms. Methods: The retinal I/R model was constructed in mice and pyroptotic markers were measured at six, 12, 24, 48, and 72 hours after I/R injury to determine the peak of pyroptotic activity. The effects of resveratrol on pyroptosis, inflammasomes, and the activation of MGCs after I/R injury were observed on the retina of mice. Moreover, induction of pyroptosis in rat Müller glial cells (r-MC) via lipopolysaccharide was used to explore the effects of resveratrol on pyroptosis of r-MC in vitro. Results: After the induction of retinal I/R injury in mice, the intricate involvement of pyroptosis in the progressive degeneration of the retina was observed, reaching its zenith at the onset of 24 hours after I/R injury. Resveratrol treatment alleviated I/R injury on the retina, relieved retinal ganglion cells death. In addition, resveratrol inhibited Caspase-1 activation, gasdermin D (GSDMD-N) cleavage, the inflammasome assembly, and the release of inflammatory cytokines, simultaneously relieving the MGCs activation. Furthermore, resveratrol inhibited the pyroptosis-related NLRP3/GSDMD-N/TMS1/ASC/Caspase-1/IL-1ß pathway in r-MC cells, and mitigated cells death in vitro. Conclusions: Pyroptosis plays an important role in the pathogenesis of retinal I/R injury. Resveratrol can attenuate pyroptotic-driven damage in the retina and MGC by inhibiting the NLRP3/GSDMD-N/TMS1/ASC/Caspase-1/IL-1ß pyroptosis pathway.

Multifunctional agents against Alzheimer's disease based on oxidative stress: Polysubstituted pyrazine derivatives synthesized by multicomponent reactions.

As Alzheimer's disease (AD) is a neurodegenerative disease with a complex pathogenesis, the exploration of multi-target drugs may be an effective strategy for AD treatment. Multifunctional small molecular agents can be obtained by connecting two or more active drugs or privileged pharmacophores by multicomponent reactions (MCRs). In this paper, two series of polysubstituted pyrazine derivatives with multifunctional moieties were designed as anti-AD agents and synthesized by Passerini-3CR and Ugi-4CR. Since the oxidative stress plays an important role in the pathological process of AD, the antioxidant activities of the newly synthesized compounds were first evaluated. Subsequently, selected active compounds were further screened in a series of AD-related bioassays, including Aß1-42 self-aggregation and deaggregation, BACE-1 inhibition, metal chelation, and protection of SH-SY5Y cells from H2O2-induced oxidative damage. Compound A3B3C1 represented the best one with multifunctional potencies. Mechanism study showed that A3B3C1 acted on Nrf2/ARE signaling pathway, thus increasing the expression of related antioxidant proteins NQO1 and HO-1 to normal cell level. Furthermore, A3B3C1 showed good in vitro human plasma and liver microsome stability, indicating a potential for further development as multifunctional anti-AD agent.

Recent advances in novel mutation genes of Parkinson's disease.

With increasing life expectancy, a growing number of individuals are being affected by Parkinson's Disease (PD), a Neurodegenerative Disease (ND). Approximately, 5-10% of PD is explained by genetic causes linked to known PD genes. With improvements in genetic testing and high-throughput technologies, more PD-associated susceptibility genes have been reported in recent years. However, a comprehensive review of the pathogenic mechanisms and physiological roles of these genes is still lacking. This article reviews novel genes with putative or confirmed pathogenic mutations in PD reported since 2019, summarizes the physiological functions and potential associations with PD. Newly reported PD-related genes include ANK2, DNAH1, STAB1, NOTCH2NLC, UQCRC1, ATP10B, TFG, CHMP1A, GIPC1, KIF21B, KIF24, SLC25A39, SPTBN1 and TOMM22. However, the evidence for pathogenic effects of many of these genes is inconclusive. A variety of novel PD-associated genes have been identified through clinical cases of PD patients and analysis of Genome-Wide Association Studies (GWAS). However, more evidence is needed in confirm the strong association of novel genes with disease.

YIPF5 (p.W218R) mutation induced primary microcephaly in rabbits.

Primary microcephaly (PMCPH) is a rare autosomal recessive neurodevelopmental disorder with a global prevalence of PMCPH ranging from 0.0013% to 0.15%. Recently, a homozygous missense mutation in YIPF5 (p.W218R) was identified as a causative mutation of severe microcephaly. In this study, we constructed a rabbit PMCPH model harboring YIPF5 (p.W218R) mutation using SpRY-ABEmax mediated base substitution, which precisely recapitulated the typical symptoms of human PMCPH. Compared with wild-type controls, the mutant rabbits exhibited stunted growth, reduced head circumference, altered motor ability, and decreased survival rates. Further investigation based on model rabbit elucidated that altered YIPF5 function in cortical neurons could lead to endoplasmic reticulum stress and neurodevelopmental disorders, interference of the generation of apical progenitors (APs), the first generation of progenitors in the developing cortex. Furthermore, these YIPF5-mutant rabbits support a correlation between unfolded protein responses (UPR) induced by endoplasmic reticulum stress (ERS), and the development of PMCPH, thus providing a new perspective on the role of YIPF5 in human brain development and a theoretical basis for the differential diagnosis and clinical treatment of PMCPH. To our knowledge, this is the first gene-edited rabbit model of PMCPH. The model better mimics the clinical features of human microcephaly than the traditional mouse models. Hence, it provides great potential for understanding the pathogenesis and developing novel diagnostic and therapeutic approaches for PMCPH.

Selective extended dissection for pancreaticoduodenectomy is associated with better survival in pancreatic cancer patients: retrospective cohort study.

BACKGROUND: Pancreatic ductal adenocarcinoma is a highly malignant tumor with relatively poor survival. Surgery is the first choice for treating patients with early pancreatic cancer. However, the surgical approach and the extent of resection for patients with pancreatic cancer are currently controversial. METHODS: The authors optimized the procedure of standard pancreaticoduodenectomy to selective extended dissection (SED), which is based on the extrapancreatic nerve plexus potentially invaded by the tumor. The authors retrospectively analyzed the clinicopathological data of patients with pancreatic adenocarcinoma who underwent radical surgery in our center from 2011 to 2020. Patients who underwent standard dissection (SD) were matched 2:1 to those who underwent SED using propensity score matching. The log-rank test and Cox regression model were used to analyze survival data. In addition, statistical analyses were performed for the perioperative complications, postoperative pathology, and recurrence pattern. RESULTS: A total of 520 patients were included in the analysis. Among patients with extrapancreatic perineural invasion (EPNI), disease-free survival was significantly longer in those who received SED than in those who received SD (14.5 months vs. 10 months, P <0.05). The incidence of metastasis in No. 9 and No. 14 lymph nodes was significantly higher in patients with EPNI. In addition, there was no significant difference in the incidence rate of perioperative complications between the two surgical procedures. CONCLUSION: Compared with SD, SED exhibits a significant prognostic benefit for patients with EPNI. The SED procedure aiming at specific nerve plexus dissection displayed particular efficacy and safety in resectable pancreatic ductal adenocarcinoma patients.

Rapamycin suppresses neuroinflammation and protects retinal ganglion cell loss after optic nerve crush.

Pyroptosis, an inflammasome-mediated mode of death, plays an important role in glaucoma. It has been shown that regulating the mTOR pathway can inhibit pyroptosis. Unfortunately, whether rapamycin (RAPA), a specific inhibitor of the mTOR pathway, can inhibit optic nerve crush (ONC)-induced pyroptosis to protect retinal ganglion cells (RGCs) has not been investigated. Our research aimed to confirm the effect of intravitreal injection of RAPA on RGCs. Furthermore, we used the ONC model to explore the underlying mechanisms. First, we observed that intravitreal injection of RAPA alleviated RGC damage induced by various types of injury. We then used the ONC model to further explore the potential mechanism of RAPA. Mechanistically, RAPA not only reduced the activation of glial cells in the retina but also inhibited retinal pyroptosis-induced expression of inflammatory factors such as nucleotide-binding oligomeric domain-like receptor 3 (NLRP3), apoptosis-associated speckle-like protein containing a CARD (ASC), N-terminal of gasdermin-D (GSDMD-N), IL-18 and IL-1ß. Moreover, RAPA exerted protective effects on RGC axons, possibly by inhibiting glial activation and regulating the mTOR/ROCK pathway. Therefore, this study demonstrates a novel mechanism by which RAPA protects against glaucoma and provides further evidence for its application in preclinical studies.

Rapid discovery and crystallography study of highly potent and selective butylcholinesterase inhibitors based on oxime-containing libraries and conformational restriction strategies.

Butyrylcholinesterase is regarded as a promising drug target in advanced Alzheimer's disease. In order to identify highly selective and potent BuChE inhibitors, a 53-membered compound library was constructed via the oxime-based tethering approach based on microscale synthesis. Although A2Q17 and A3Q12 exhibited higher BuChE selectivity versus acetylcholinesterase, the inhibitory activities were unsatisfactory and A3Q12 did not inhibit Aß1-42 peptide self-induced aggregation. With A2Q17 and A3Q12 as leads, a novel series of tacrine derivatives with nitrogen-containing heterocycles were designed based on conformation restriction strategy. The results demonstrated that 39 (IC50 = 3.49 nM) and 43 (IC50 = 7.44 nM) yielded much improved hBuChE inhibitory activity compared to the lead A3Q12 (IC50 = 63 nM). Besides, the selectivity indexes (SI = AChE IC50 / BChE IC50) of 39 (SI = 33) and 43 (SI = 20) were also higher than A3Q12 (SI = 14). The results of the kinetic study showed that 39 and 43 exhibited a mixed-type inhibition against eqBuChE with respective Ki values of 1.715 nM and 0.781 nM. And 39 and 43 could inhibit Aß1-42 peptide self-induced aggregation into fibril. X-ray crystallography structures of 39 or 43 complexes with BuChE revealed the molecular basis for their high potency. Thus, 39 and 43 are deserve for further study to develop potential drug candidates for the treatment of Alzheimer's disease.

Contributions of NR1H3 genetic polymorphisms to susceptibility and effects of narrowband UVB phototherapy to nonsegmental vitiligo.

Vitiligo is the most common depigmenting disorder to which both genetic and environmental factors contribute. The aim of the current work was to evaluate the relationship between polymorphisms of the gene nuclear receptor subfamily 1 Group H member 3 (NR1H3) and the risk of vitiligo and phototherapy effects in the Chinese Han population. Two independent samples were enrolled to form the discovery set (comprised of 1668 nonsegmental vitiligo [NSV] patients and 2542 controls) and the validation set (comprised of 745 NSV patients and 1492 controls). A total of 13 tag single nucleotide polymorphisms (SNPs) were genotyped in the samples from the discovery stage. SNPs that achieved nominal significance were validated in another independent sample set. The serum level of NR1H3 protein was assayed using enzyme-linked immunosorbent assay kits in the validation set. Genetic association analysis was carried out at allelic and genotypic levels. The therapeutic effects of significant SNPs were examined in the validation set. The SNP rs3758672 was significantly associated with NSV. The A allele was correlated with NSV risk and poorer therapeutic effects. The A allele was strongly correlated with the increased level of serum NR1H3 in both controls and patients. In summary, SNP rs3758672 in NR1H3 was significantly associated with both disease susceptibility and individualized therapeutic effects of NSV in study participants with Han Chinese ancestry.

Clinical and genetic findings in a Chinese cohort with choroideremia.

OBJECTIVE: Choroideremia (CHM) is an X-linked chorioretinal dystrophy caused by variants in the CHM gene. The aim of this study was to report the clinical and genetic features of a cohort of affected males with CHM and establish the relationship between best correct visual acuity (BCVA) and age. METHOD: Twenty-seven patients from 24 unrelated families underwent detailed ophthalmic examinations and comprehensive molecular genetic analysis. We combined the 27 patients in our own cohort with 68 Chinese patients from six previously reported studies to determine a transition age for BCVA rapid decline in 95 patients. RESULTS: Twenty-three causal (9 novel) CHM variants were identified in the 27 patients, who had a mean age of 30.5 ± 17.4 years and a mean BCVA (LogMAR) of 0.61 ± 0.79. Patients at different disease stages showed different extents of retinal pigment epithelium (RPE) and choroid abnormalities. Central retinal optical coherence tomography (OCT) scanning revealed defects in the ellipsoid zone and RPE in all patients and outer retinal tubulations in 75%. The 95 patients had a mean age of 33.27 ± 16.27 years and an average (LogMAR) of 0.72 ± 0.82. The BCVA did not decline rapidly before age 25, but decreased at a mean rate of 0.037logMAR/year after that age. CONCLUSIONS: Our results indicated Chinese patients with CHM variants have a younger transition age for rapid BCVA decline than previously reported for other ethnic groups. Central retinal OCT scanning can identify different abnormalities in the retinal structures, and these might be used as other parameters for monitoring disease progression in patients with CHM.

Current situation and progress of drugs for reducing intraocular pressure.

Glaucoma, the most common cause of irreversible blindness worldwide, usually causes characteristic optic nerve damage. Pathological intraocular pressure (IOP) elevation is a major risk factor. Drug reduction of IOP is the preferred treatment for clinicians because it can delay the progression of disease. However, the traditional IOP-lowering drugs currently used by patients may be poorly tolerated. Therefore, in recent years, some new drugs have been put into clinical application or in clinical phase I-III studies. They have a better IOP-lowering effect and fewer adverse reactions. Because glaucoma is a chronic disease, drugs need to be administered continuously for a long time. For patients, good compliance and high drug bioavailability have a positive effect on the prognosis of the disease. Therefore, clinicians and scientists have developed drug delivery systems to solve this complex problem. In addition, natural compounds and dietary supplements have a good effect of reducing IOP, and they can also protect the optic nerve through antioxidant action. We summarize the current traditional drugs, new drugs, sustained-release drug delivery systems, and complementary drugs and outline the mechanism of action and clinical effects of these drugs on glaucoma and their recent advances.

CRISPR/Cas9-mediated deletion of Fam83h induces defective tooth mineralization and hair development in rabbits.

Family with sequence similarity 83 members H (Fam83h) is essential for dental enamel formation. Fam83h mutations cause human amelogenesis imperfecta (AI), an inherited disorder characterized by severe hardness defects in dental enamel. Nevertheless, previous studies showed no enamel defects in Fam83h-knockout/lacZ-knockin mice. In this study, a large deletion of the Fam83h gene (900 bp) was generated via a dual sgRNA-directed CRISPR/Cas9 system in rabbits. Abnormal tooth mineralization and loose dentine were found in homozygous Fam83h knockout (Fam83h-/- ) rabbits compared with WT rabbits. In addition, reduced hair follicle counts in dorsal skin, hair cycling dysfunction and hair shaft differentiation deficiency were observed in Fam83h-/- rabbits. Moreover, X-rays and staining of bone sections showed abnormal bending of the ulna and radius and an ulnar articular surface with insufficient trabecular bone in Fam83h-/- rabbits. Taken together, these data are the first report of defective hair cycling, hair shaft differentiation and abnormal bending of the ulna and radius in Fam83h-/- rabbits. This novel Fam83h-/- rabbit model may facilitate understanding the function of Fam83h and the pathogenic mechanism of the Fam83h mutation.

Phenotype-Based Genetic Analysis Reveals Missing Heritability of ABCA4-Related Retinopathy: Deep Intronic Variants and Copy Number Variations.

Purpose: To identify the missing heritability of ABCA4-related retinopathy in a Chinese cohort. Methods: We recruited 33 unrelated patients with ABCA4-related retinopathy carrying a monoallelic variant in ABCA4. All patients underwent ophthalmic examinations. Next-generation sequencing of the whole ABCA4 sequence, including coding and noncoding regions, was performed to detect deep intronic variants (DIVs) and copy number variations (CNVs). Results: We identified eight missing pathogenic ABCA4 variants in 60.6% of the patients (20/33), which comprised five DIVs and three CNVs. The five DIVs, including four novel (c.1555-816T>G, c.2919-169T>G, c.2919-884G>T, and c.5461-1321A>G) and one reported (c.4539+1100A>G), accounted for the missing alleles in 51.5% of the patients. Minigene assays showed that four novel DIVs activated cryptic splice sites leading to the insertions of pseudoexons. The three novel CNVs consisted of one gross deletion of 1273 bp (exon 2) and two gross duplications covering 25.2 kb (exons 28-43) and 9.4 kb (exons 38-44). The microhomology domains were identified at the breakpoints and revealed the potential mechanisms of CNV formation. Conclusions: DIVs and CNVs explained approximately two-thirds of the unresolved Chinese cases with ABCA4-related retinopathy. Combining results from phenotypic-directed screening, targeting the whole ABCA4 sequencing and in silico tools can help to identify the missing heritability.