Exome and genome sequencing to unravel the precise breakpoints of partial trisomy 6q and partial Monosomy 2q.

BACKGROUND: Patients with complex phenotypes and a chromosomal translocation are particularly challenging, since several potentially pathogenic mechanisms need to be investigated. CASE PRESENTATION: Here, we combined exome and genome sequencing techniques to identify the precise breakpoints of heterozygous microduplications in the 6q25.3-q27 region and microdeletions in the 2q37.1-q37.3 region in a proband. The 5-year-old girl exhibited a severe form of congenital cranial dysinnervation disorder (CCDD) in addition to skeletal dysmorphism anomalies and severe intellectual disability. This is the second case affecting chromosomes 2q and 6q. The individual's karyotype showed an unbalanced translocation 46,XX,del(2)t(2;6)(q37.1;q25.3), which was inherited from her unaffected father [46,XY,t(2;6)(q37.1;q25.3)]. We also obtained the precise breakpoints of a de novo heterozygous copy number deletion [del(2)(q37.1q37.3)chr2:g.232963568_24305260del] and a copy number duplication [dup(6)(q25.3q27)chr6:g.158730978_170930050dup]. The parental origin of the observed balanced translocation was not clear because the parents declined genetic testing. CONCLUSION: Patients with a 2q37 deletion and 6q25.3 duplication may exhibit severe significant neurological and skeletal dysmorphisms, and the utilization of exome and genome sequencing techniques has the potential to unveil the entire translocation of the CNV and the precise breakpoint.

Screening of genes interacting with high myopia and neuropsychiatric disorders.

Clinical studies have demonstrated an association between high myopia (HM) and neuropsychiatric disorders; however, the underlying mechanism of the association is not clear. We used whole exome sequencing (WES) in combination with the Genetic Variants Classification Criteria and Guidelines published by the American College of Medical Genetics (ACMG) and bioinformatics analysis to clarify the interrelationship between candidate genes. Causative genes for ocular diseases (45.38%) followed by neuropsychiatric disorders (22.69%) accounted for the highest proportion of genes that exhibited high pathogenicity in HM patients were found. Four pathogenic gene mutations were identified according to ACMG guidelines: c.164_165insACAGCA and c.C1760T in POLG, c.G1291A in COL5A1, and c.G10242T in ZNF469. Three causative genes for neuropsychiatric diseases, PTPRN2, PCDH15 and CDH23, were found to fall at the HM locus. The above results suggest that these genes may interact in high myopia and neuropsychiatric diseases.

Tacrolimus ameliorates bleomycin-induced pulmonary fibrosis by inhibiting M2 macrophage polarization via JAK2/STAT3 signaling.

Idiopathic pulmonary fibrosis (IPF) is a progressive fibrotic lung disease of unknown cause and characterized by excessive proliferation of fibroblasts and the irregular remodeling of extracellular matrix (ECM), which ultimately cause the severe distortion of the alveolar architecture. The median survival of IPF patients is 2-5 years. IPF patients are predominantly infiltrated by M2 macrophages during the course of disease development and progression. Predominantly accumulation of M2 macrophages accelerates fibrosis progression by secreting multiple cytokines that promote fibroblast to myofibroblast transition. In the process of M2 macrophage polarization, JAK2/STAT3 signaling plays a key role, thus, targeting activated macrophages to inhibit the pro-fibrotic phenotype is considered as an approach to the potential treatment of IPF. Tacrolimus is a macrolide antibiotic that as a specific inhibitor of T-lymphocyte function and has been used widely as an immunosuppressant in human organ transplantation. In this study we explored the potential effect and mechanism of tacrolimus on pulmonary fibrosis in vivo and vitro. Here, we found that tacrolimus is capable of suppressing M2 macrophages polarization by inhibiting pro-fibrotic factors secreted by M2 macrophages. This effect further alleviates M2-induced myofibroblast activation, thus resulting in a decline of collagen deposition, pro-fibrotic cytokines secretion, recovering of lung function, ultimately relieving the progression of fibrosis in vivo. Mechanistically, we found that tacrolimus can inhibit the activation of JAK2/STAT3 signaling by targeting JAK2. Our findings indicate a potential anti-fibrotic effect of tacrolimus by regulating macrophage polarization and might be meaningful in clinical settings.

Novel Compound Heterozygous Variations in MPDZ Gene Caused Isolated Bilateral Macular Coloboma in a Chinese Family.

Macular coloboma (MC) is a rare congenital retinochoroidal defect characterized by lesions of different sizes in the macular region. The pathological mechanism underlying congenital MC is unknown. Novel compound heterozygous variations, c.4301delA (p.Asp1434fs*3) and c.5255C>G (p.Ser1752Ter), in the multiple PDZ domain (MPDZ) proteins were identified via whole-exome analysis on the proband with isolated bilateral macular coloboma in a Chinese family. Segregation analysis revealed that each of the unaffected parents was heterozygous for one of the two variants. The results of the in silico and bioinformatics analysis were aligned with the experimental data. The knockdown of MPDZ in zebrafish caused a decrease in the ellipsoid zone, a destruction of the outer limiting membrane, and the subsequent RPE degeneration. Overall, the loss of MPDZ in zebrafish contributed to retinal development failure. These results indicate that MPDZ plays an essential role in the occurrence and maintenance of the macula, and the novel compound heterozygous variations were responsible for an autosomal recessive macular deficiency in this Chinese family.

PKM2 promotes pulmonary fibrosis by stabilizing TGF-ß1 receptor I and enhancing TGF-ß1 signaling.

Idiopathic pulmonary fibrosis (IPF) is a progressive interstitial lung disease, and the molecular mechanisms remain poorly understood. Our findings demonstrated that pyruvate kinase M2 (PKM2) promoted fibrosis progression by directly interacting with Smad7 and reinforcing transforming growth factor-ß1 (TGF-ß1) signaling. Total PKM2 expression and the portion of the tetrameric form elevated in lungs and fibroblasts were derived from mice with bleomycin (BLM)-induced pulmonary fibrosis. Pkm2 deletion markedly alleviated BLM-induced fibrosis progression, myofibroblast differentiation, and TGF-ß1 signaling activation. Further study showed that PKM2 tetramer enhanced TGF-ß1 signaling by directly binding with Smad7 on its MH2 domain, and thus interfered with the interaction between Smad7 and TGF-ß type I receptor (TßR1), decreased TßR1 ubiquitination, and stabilized TßR1. Pharmacologically enhanced PKM2 tetramer by TEPP-46 promoted BLM-induced pulmonary fibrosis, while tetramer disruption by compound 3k alleviated fibrosis progression. Our results demonstrate how PKM2 regulates TGF-ß1 signaling and is a key factor in fibrosis progression.

Lenalidomide attenuates post-inflammation pulmonary fibrosis through blocking NF-κB signaling pathway.

Idiopathic pulmonary fibrosis (IPF) is a pathological consequence of interstitial pulmonary diseases, and is characterized by the persistence of fibroblasts and excessive deposition of extracellular matrix (ECM). The etiology of IPF is multifactorial. Although the role of inflammation in fibrogenesis is controversial, it is still recognized as an important component and epiphenomenon of IPF. Stimulus increase production of pro-inflammatory cytokines and activation of NF-κB, which will further promote inflammation response and myofibroblast transition. Lenalidomide is an immunomodulatory drug. Previous studies have revealed its anti-tumor effects through regulating immune response. Here we investigate the effect of lenalidomide on post-inflammation fibrosis. In vitro study revealed that lenalidomide inhibited NF-κB signaling in LPS-induced macrophage, and further attenuated macrophage-induced myofibroblast activation. Meanwhile, lenalidomide could inhibit TGF-ß1-induced myofibroblast activation through suppressing TGF-ß1 downstream MAPK signaling. In vivo study showed that lenalidomide inhibited pro-inflammatory cytokines TNF-α and IL-6 while enhanced anti-fibrotic cytokines IFN-γ and IL-10 in bleomycin-induced inflammation model, and attenuated pulmonary fibrosis and collagen deposition in the following fibrosis stage. In conclusion, our results demonstrate that lenalidomide possesses potential anti-fibrotic effects through suppressing NF-κB signaling.

Clevudine attenuates bleomycin-induced early pulmonary fibrosis via regulating M2 macrophage polarization.

Pulmonary fibrosis (IPF) is a chronic, progressive interstitial lung disease. It is a growing clinical problem which can result in breathlessness or respiratory failure and has an average life expectancy of 3 years from diagnosis. Predominantly accumulation of M2 macrophages accelerates fibrosis progression by secreting multiple cytokines that promote fibroblast to myofibroblast transition and aberrant wound healing of epithelial cells. Targeting activated macrophages to inhibit the pro-fibrotic phenotype is considered as an approach for the potential treatment of PF. Clevudine is s a purine nucleoside analogue which in an oral formulation is approved for treatment of patients with hepatitis B virus (HBV). Here, we found that clevudine is capable of suppressing pro-fibrotic phenotype (i.e., CD206, Arg1 and YM1) of M2 macrophages while enhancing anti-fibrotic phenotype (i.e., CD86, IL-6 and IL-10) by inhibiting PI3K/Akt signaling pathway. This effect further alleviates M2-induced myofibroblast activation and epithelial-to-mesenchymal transition (EMT), thus resulting in a decline of collagen deposition, pro-fibrotic cytokines secretion, with a concomitant recover ofpulmonary functions in vivo. Less infiltration of M2 macrophages between α-SMA + cells was also found in clevudine treated mice. Our findings indicate a potential anti-fibrotic effect of clevudine by regulating macrophage polarization and might be meaningful in clinical settings.

Fedratinib Attenuates Bleomycin-Induced Pulmonary Fibrosis via the JAK2/STAT3 and TGF-ß1 Signaling Pathway.

Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive interstitial lung disease with multiple causes, characterized by excessive myofibrocyte aggregation and extracellular matrix deposition. Related studies have shown that transforming growth factor-ß1 (TGF-ß1) is a key cytokine causing fibrosis, promoting abnormal epithelial-mesenchymal communication and fibroblast-to-myofibroblast transition. Fedratinib (Fed) is a marketed drug for the treatment of primary and secondary myelofibrosis, targeting selective JAK2 tyrosine kinase inhibitors. However, its role in pulmonary fibrosis remains unclear. In this study, we investigated the potential effects and mechanisms of Fed on pulmonary fibrosis in vitro and in vivo. In vitro studies have shown that Fed attenuates TGF-ß1- and IL-6-induced myofibroblast activation and inflammatory response by regulating the JAK2/STAT3 signaling pathway. In vivo studies have shown that Fed can reduce bleomycin-induced inflammation and collagen deposition and improve lung function. In conclusion, Fed inhibited inflammation and fibrosis processes induced by TGF-ß1 and IL-6 by targeting the JAK2 receptor.

Comparison of clinical efficacy of metoprolol combined with irbesartan and hydrochlorothiazide and non-invasive ventilator in the emergency treatment of patients with severe heart failure.

Clinical efficacy of metoprolol combined with irbesartan and hydrochlorothiazide and non-invasive ventilator in the emergency treatment of patients with severe heart failure (HF) was investigated. A retrospective analysis of the medical records of 124 patients with severe HF admitted to Binzhou Medical University Hospital from May 2012 to August 2016 was performed. Among them, 78 patients who were treated with metoprolol combined with irbesartan and hydrochlorothiazide and non-invasive ventilator for emergency treatment were enrolled into the Research Group, while the Control Group consisted of 46 patients treated with routine medical treatment. Echocardiographic parameters, 6-minute walking test results, the efficacy of the emergency treatment, cardiac function grading, left ventricular ejection fraction (LVEF) and brain natriuretic peptide (BNP) levels were compared between the two groups. After the emergency treatment, the echocardiographic indexes of both groups increased to different extents (P<0.05). Compared with the results before, patients of both groups walked longer within six minutes after the emergency treatment (P<0.05). After the emergency treatment, the BNP expression levels in the two groups decreased to different degrees (P<0.05). After a 7-day emergency treatment, the efficiency rate of treatment of the Research Group was higher than that of the Control Group (P<0.05). The method is helpful for the recovery of respiratory function, for relieving symptoms in short time, improving cardiac function and promising high safety, using metoprolol and irbesartan and hydrochlorothiazide combined with non-invasive ventilator had satisfactory clinical efficacy in the emergency treatment of patients with severe heart failure and is thus worthy of clinical promotion.

Synthesis of Acyl Azides from 1,3-Diketones via Oxidative Cleavage of Two C-C Bonds.

A metal-free PhI(OAc)2-mediated method for the synthesis of acyl azides through oxidative cleavage of 1,3-diketones is described. This method is shown to have a broad substrate scope, providing a useful tool for multiproduct synthesis in a single procedure. A possible reaction pathway is proposed based on mechanistic studies.

Pathway­based detection of idiopathic pulmonary fibrosis at an early stage.

Idiopathic pulmonary fibrosis (IPF) is the most common interstitial pneumonia and the most aggressive interstitial lung disease. Usually, IPF is confirmed by the histopathological pattern of typical interstitial pneumonia and requires an integrated multidisciplinary approach from pulmonologists, radiologists and pathologists. However, these diagnoses are performed at an advanced stage of IPF. At present, pathway­based detection requires investigation, as it can be performed at an early stage of the disease. The aim of the present study was to find an effective method of diagnosing IPF at an early stage. Microarray data forE­GEOD­33566 were downloaded from the ArrayExpress database. Human pathways were downloaded from Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway database. An individual pathway­based method to diagnose IPF at an early stage was introduced. Pathway statistics were analyzed with an individualized pathway aberrance score. P­values were obtained with different methods, including the Wilcoxon test, linear models for microarray data (Limma) test and attract methods, generating three pathway groups. Support vector machines (SVM) were used to identify the best group for diagnosing IPF at an early stage. There were 106 differential pathways in Wilcoxon­based KEGG Pathway (n>5) group, 100 in the Limma­based KEGG Pathway (n>5) group, and seven in the attract­based KEGG Pathway (n>5) group. The pathway statistics of these differential pathways in three groups were analyzed with linear SVM. The results demonstrated that the Wilcoxon­based KEGG Pathway (n>5) group performed best in diagnosing IPF.

CEP78 is mutated in a distinct type of Usher syndrome.

BACKGROUND: Usher syndrome is a genetically heterogeneous disorder featured by combined visual impairment and hearing loss. Despite a dozen of genes involved in Usher syndrome having been identified, the genetic basis remains unknown in 20-30% of patients. In this study, we aimed to identify the novel disease-causing gene of a distinct subtype of Usher syndrome. METHODS: Ophthalmic examinations and hearing tests were performed on patients with Usher syndrome in two consanguineous families. Target capture sequencing was initially performed to screen causative mutations in known retinal disease-causing loci. Whole exome sequencing (WES) and whole genome sequencing (WGS) were applied for identifying novel disease-causing genes. RT-PCR and Sanger sequencing were performed to evaluate the splicing-altering effect of identified CEP78 variants. RESULTS: Patients from the two independent families show a mild Usher syndrome phenotype featured by juvenile or adult-onset cone-rod dystrophy and sensorineural hearing loss. WES and WGS identified two homozygous rare variants that affect mRNA splicing of a ciliary gene CEP78. RT-PCR confirmed that the two variants indeed lead to abnormal splicing, resulting in premature stop of protein translation due to frameshift. CONCLUSIONS: Our results provide evidence that CEP78 is a novel disease-causing gene for Usher syndrome, demonstrating an additional link between ciliopathy and Usher protein network in photoreceptor cells and inner ear hair cells.

FGFR2 mutation in a Chinese family with unusual Crouzon syndrome.

AIM: To describe the clinical characteristics with genetic lesions in a Chinese family with Crouzon syndrome. METHODS: All five patients from this family were included and received comprehensive ophthalmic and systemic examinations. Direct sequencing of the FGFR2 gene was employed for mutation identification. Crystal structure analysis was applied to analyze the structural changes associated with the substitution. RESULTS: All patients presented typical Crouzon features, including short stature, craniosynostosis, mandibular prognathism, shallow orbits with proptosis, and exotropia. Intrafamilial phenotypic diversities were observed. Atrophic optic nerves were exclusively detected in the proband and her son. Cranial magnetic resonance imaging (MRI) implied a cystic lesion in her sellar and third ventricular regions. A missense mutation, FGFR2 p.Cys342Trp, was found as disease causative. This substitution would generate conformational changes in the extracellular Ig-III domain of the FGFR-2 protein, thus altering its physical and biological properties. CONCLUSION: We describe the clinical presentations and genotypic lesions in a Chinese family with Crouzon syndrome. The intrafamilial phenotypic varieties in this family suggest that other genetic modifiers may also play a role in the pathogenesis of Crouzon syndrome.

Whole exome sequencing confirms the clinical diagnosis of Marfan syndrome combined with X-linked hypophosphatemia.

BACKGROUND: To determine the genetic lesions and to modify the clinical diagnosis for a Chinese family with significant intrafamilial phenotypic diversities and unusual presentations. METHODS: Three affected patients and the asymptomatic father were included and received comprehensive systemic examinations. Whole exome sequencing (WES) was performed for mutation detection. Structural modeling test was applied to analyze the potential structural changes caused by the missense substitution. RESULTS: The proband showed a wide spectrum of systemic anomalies, including bilateral ectopia lentis, atrial septal defect, ventricular septal defect, widening of tibial metaphysis with medial bowing, and dolichostenomelia in digits, while her mother and elder brother only demonstrated similar skeletal changes. A recurrent mutation, PHEX p.R291*, was found in all patients, while a de novo mutation, FBN1 p.C792F, was only detected in the proband. The FBN1 substitution was also predicted to cause significant conformational change in fibrillin-1 protein, thus changing its physical and biological properties. CONCLUSIONS: Taken together, we finalized the diagnosis for this family as X-linked hypophosphatemia (XLH), and diagnosed this girl as Marfan syndrome combined with XLH, and congenital heart disease. Our study also emphasizes the importance of WES in assisting the clinical diagnosis for complicated cases when the original diagnoses are challenged.

Identification of a novel p.R1443W mutation in RP1 gene associated with retinitis pigmentosa sine pigmento.

AIM: To screen mutations in the retinitis pigmentosa 1 (RP1) gene and the rhodopsin (RHO) gene in Chinese patients with retinitis pigmentosa sine pigmento (RPSP) and describe the genotype-phenotype relationship of the mutations. METHODS: Twenty affected, unrelated Chinese individuals with RPSP (4 autosomal dominant RPSP, 12 autosomal recessive RPSP and 4 unknown inheritance pattern) were recruited between 2009 and 2012. The clinical features were determined by complete ophthalmologic examinations. Polymerase chain reaction (PCR) and direct DNA sequencing were used to screen the entire coding region and splice junctions of the RP1 gene and the RHO gene. The cosegregation analysis and population frequency studies were performed for patients with identified mutations. RESULTS: Five variants in the RP1 gene and one in the RHO gene were detected in 20 probands. Four missense changes (rs444772, rs446227, rs414352, rs441800) and one non-coding variant (rs56340615) were common SNPs and none of them showed a significant relationship with RPSP. A missense mutation p.R1443W was identified in the RP1 gene in three affected individuals from a family with autosomal dominant RPSP and was found to cosegregate with the phenotype in this family, suggestive of pathogenic. In addition, population frequency analysis showed the p.R1443W mutation was absent in 300 healthy controls. CONCLUSION: The identification of p.R1443W mutation cosegregating in a family with autosomal dominant RPSP highlights an atypical phenotype of the RP1 gene mutation, while RHO gene is not associated with the pathogenesis of RPSP in this study. To our knowledge, this is the fist mutation identified to associate with RPSP.

Association of insulin-like growth factor-1 polymorphisms with high myopia in the Chinese population.

PURPOSE: The purpose of this study was to determine whether genetic variants in the insulin-like growth factor-1 (IGF-1) gene were associated with high myopia in the Chinese population. METHODS: A case-control association study of 421 unrelated Chinese patients with high myopia and 401 control subjects matched in ethnicity and gender was undertaken. Genomic DNA was prepared from peripheral blood. All individuals were genotyped for 7 tag single nucleotide polymorphisms (tSNPs) across the IGF-1 gene region. Genotypic distribution was tested for Hardy-Weinberg equilibrium. The genotype and allele frequencies were evaluated using the χ(2) tests. Bonferroni corrections for multiple comparisons were performed. RESULTS: The polymorphism of rs12423791 showed positive association with extreme myopia (p(allel)=0.006 and p(allel1 recessive model)=0.004, respectively) after Bonferroni correction for multiple testing and the haplotype GC of rs5742629-rs12423791 was also associated with extreme myopia (p=0.033) after 50,000 permutations for multiple comparisons. CONCLUSIONS: The polymorphism of rs12423791 in IGF-1 may be associated with extreme myopia in the Chinese population and should be investigated further.