Improving the storage quality and suppressing off-flavor generation of winter jujube by precise micro-perforated MAP.

Introduction: Traditional modified atmosphere packaging (MAP) cannot meet the preservation requirements of winter jujube, and the high respiration rate characteristics of winter jujube will produce an atmosphere component with high CO2 concentration in traditional MAP. Micro-perforated MAP is suitable for the preservation of winter jujube due to its high permeability, which can effectively remove excess CO2 and supply O2. In this study, a microporous film preservation system that can be quickly applied to winter jujube was developed, namely PMP-MAP (precise micro-perforated modified atmosphere packaging). An experiment was designed to store winter jujube in PMP-MAP at 20°C and 2°C, respectively. The quality, aroma and antioxidant capacity, etc. of winter jujube at the storage time were determined. Methods: In this study, the optimal micropore area required for microporous film packaging at different temperatures is first determined. To ensure the best perforation effect, the effects of various factors on perforation efficiency were studied. The gas composition within the package was predicted using the gas prediction equation to ensure that the gas composition of the perforated package achieved the desired target. Finally, storage experiments were designed to determine the quality index of winter jujube, including firmness, total soluble solids, titratable acid, reddening, and decay incidence. In addition, sensory evaluation, aroma and antioxidant capacity were also determined. Finally, the preservation effect of PMP-MAP for winter jujube was evaluated by combining the above indicators. Results and discussion: At the end of storage, PMP-MAP reduced the respiration rate of winter jujube, which contributed to the preservation of high total soluble solids and titratable acid levels, and delayed the reddening and decay rate of winter jujube. In addition, PMP-MAP maintained the antioxidant capacity and flavor of winter jujube while inhibiting the occurrence of alcoholic fermentation and off-flavors. This can be attributed to the effective gas exchange facilitated by PMP-MAP, thereby preventing anaerobic stress and quality degradation. Therefore, the PMP-MAP approach is an efficient method for the storage of winter jujube.

Embryonic exposure to aluminum chloride blocks the onset of spermatogenesis through disturbing the dynamics of testicular tight junctions via upregulating Slc25a5 in offspring.

Studies have revealed neurotoxicity, hepatotoxicity, and developmental and reproductive toxicity in mice exposed to aluminum. However, relatively few studies have been conducted to clarify the mechanism underlying the impact of embryonic exposure to aluminum on the development of the male reproductive system in offspring. Pregnant mice were administered aluminum chloride (AlCl3) by gavage from day 12.5 of gestation until birth. Our findings demonstrated that embryonic exposure to AlCl3 disrupted testicular development and spermatogenesis by impairing testicular architecture, reducing sperm count, and upregulating the expression of tight junction (TJ) protein between Sertoli cells (SCs). Further in vitro studies revealed that treatment with AlCl3 stabilized TJ proteins Occludin and ZO-1 expression by inhibiting ERK signaling pathway activation, thereby upregulating Slc25a5 expression which induced ATP production leading to disruption of cytoskeletal protein homeostasis. Therefore, the study provided a new mechanistic insight into how AlCl3 exposure interfered with testicular development and spermatogenesis while suggesting that Slc25a5 might be a target affected by AlCl3 influencing cell metabolism.

Quantitative proteomics reveals PPAR signaling pathway regulates the cardiomyocyte activity of neonatal mouse heart.

Cardiovascular diseases (CVDs) are among the most morbid and deadly types of diseases worldwide, while the existing therapeutic approaches all have their limitations. Mouse heart undergoes a very complex postnatal developmental process, including the 1-week window in which cardiomyocytes (CMs) maintain relatively high cell activity. The underlying mechanism provides an attractive direction for CVDs treatment. Herein, we collected ventricular tissues from mice of different ages from E18.5D to P8W and performed iTRAQ-based quantitative proteomics to characterize the atlas of cardiac development. A total of 3422 proteins were quantified at all selected time points, revealing critical proteomic changes related to cardiac developmental events such as the metabolic transition from glycolysis to beta-oxidation. A cluster of significantly dysregulated proteins containing proteins that have already been reported to be associated with cardiac regeneration (Erbb2, Agrin, and Hmgb) was identified. Meanwhile, the peroxisome proliferator-activated receptor (PPAR) signaling pathway (Cpt1α, Hmgcs2, Plin2, and Fabp4) was also found specifically enriched. We further revealed that bezafibrate, a pan-activator of PPAR signaling pathway markedly enhanced H9C2 cardiomyocyte activity via enhancing Cpt1α expression. This work provides new hint that activation of PPAR signaling pathway could potentially be a therapeutic strategy for the treatment of CVDs.

Minigene Assay as an Effective Molecular Diagnostic Strategy in Determining the Pathogenicity of Noncanonical Splice-Site Variants in FLCN.

Primary spontaneous pneumothorax (PSP) or pulmonary cyst is one of the manifestations of Birt-Hogg-Dubé syndrome, which is caused by pathogenic variants in FLCN gene. Genetic testing in patients with PSP identifies a certain number of missense or intronic variants. These variants are usually considered as variants of uncertain significance, whose functional interpretations pose a challenge in clinical genetics. To improve recognition of pathogenic splice-altering variants in FLCN gene, computational tools are used to prioritize potential splice-altering variants and then a hybrid minigene assay is performed to verify the RNA splicing pattern. Herein, variants in FLCN exon 11 and its flanking sequence are focused. Eight variants detected in 11 patients with PSP are evaluated, and six variants are prioritized by in silico tools as potential splice-altering variants of uncertain significance. Four variants (c.1177-5_1177-3delCTC, c.1292_1300+4del, c.1300+4C>T, and c.1300+5G>A) are demonstrated by minigene assay to alter RNA splicing of FLCN, and the last three of them are novel. RT-PCR of patient-derived RNA gives consistent results. Genotype-phenotype correlation analysis in patients with PSP with these variants demonstrates good concordance. Our results underline the importance of RNA analysis, which could provide molecular evidence for pathogenicity of a variant, and provide essential information for the clinical interpretation of variants. Combining the clinical information, a definitive diagnosis could be made.

Development of functional anti-Gn nanobodies specific for SFTSV based on next-generation sequencing and proteomics.

Severe fever with thrombocytopenia syndrome (SFTS) is an acute infectious disease caused by novel bunyavirus (SFTSV), with a mortality rate of 6.3% ~ 30%. To date, there is no specific treatment for SFTS. Previously, we demonstrated that SFTSV surface glycoprotein (Glycoprotein N, Gn) was a potential target for the development of SFTS vaccine or therapeutic antibodies, and anti-Gn neutralizing antibodies played a protective role in SFTS infection. Compared with traditional antibodies, nanobodies from camelids have various advantages, including small molecular weight, high affinity, low immunogenicity, convenient production by gene engineering, etc. In this study, we combined next-generation sequencing (NGS) with proteomics technology based on affinity purification-mass spectrometry (AP-MS) and bioinformatics analysis to high-throughput screen monoclonal anti-Gn nanobodies from camel immunized with Gn protein. We identified 19 anti-Gn monoclonal nanobody sequences, of which six sequences were selected for recombinant protein expression and purification. Among these six anti-Gn nanobodies, nanobody 57,493 was validated to be highly specific for Gn. The innovative high-throughput technical route developed in this study could also be expanded to the production of nanobodies specific for other viruses like SARS-CoV-2.

E. coli Membrane Vesicles as a Catalase Carrier for Long-Term Tumor Hypoxia Relief to Enhance Radiotherapy.

Hypoxia is one of the most important factors that limit the effect of radiotherapy, and the abundant H2O2 in tumor tissues will also aggravate hypoxia-induced radiotherapy resistance. Delivering catalase to decompose H2O2 into oxygen is an effective strategy to relieve tumor hypoxia and radiotherapy resistance. However, low stability limits catalase's in vivo application, which is one of the most common limitations for almost all proteins' internal utilization. Here, we develop catalase containing E. coli membrane vesicles (EMs) with excellent protease resistance to relieve tumor hypoxia for a long time. Even treated with 100-fold of protease, EMs showed higher catalase activity than free catalase. After being injected into tumors post 12 h, EMs maintained their hypoxia relief ability while free catalase lost its activity. Our results indicate that EMs might be an excellent catalase delivery for tumor hypoxia relief. Combined with their immune stimulation features, EMs could enhance radiotherapy and induce antitumor immune memory effectively.

SS-31 efficacy in a mouse model of Friedreich ataxia by upregulation of frataxin expression.

Friedreich ataxia (FRDA) is a serious hereditary neurodegenerative disease, mostly accompanied with hypertrophic cardiomyopathy, caused by the reduced expression of frataxin (FXN). However, there is still no effective treatment. Our previous studies have shown that SS-31, a mitochondrion-targeted peptide, is capable to upregulate the expression of FXN and improve the mitochondrial function in cells derived from FRDA patients. To further explore the potential of SS-31, we used the GAA expansion-based models, including Y47 and YG8R (Fxn KIKO) mice, primary neurons and macrophages from the mice and cells derived from FRDA patients. After once-daily intraperitoneal injection of 1 mg/kg SS-31 for 1 month, we observed the significant improvement of motor function. The vacuolation in dorsal root ganglia, lesions in dentate nuclei and the lost thickness of myelin sheath of spinal cord were all repaired after SS-31 treatment. In addition, the hypertrophic cardiomyocytes and disarrayed abnormal Purkinje cells were dramatically reduced. Interestingly, we found that SS-31 treatment upregulated FXN expression not only at the translational levels as observed in cell culture but also at mRNA levels in vivo. Consequently, mitochondrial morphology and function were greatly improved in all tested tissues. Importantly, our data provided additional evidence that the maintenance of the therapeutic benefits needed continuous drug administration. Taken together, our findings have demonstrated the effectiveness of SS-31 treatment through the upregulation of FXN in vivo and offer guidance of the potential usage in the clinical application for FRDA.

Inhibition of heat shock protein (HSP) 90 reverses signal transducer and activator of transcription (STAT) 3-mediated muscle wasting in cancer cachexia mice.

BACKGROUND AND PURPOSE: Cancer cachexia is a common cause of death among cancer patients with no currently effective treatment available. In animal models, aberrant activation of STAT3 in skeletal muscle contributes to muscle wasting. However, clinically the factors regulating STAT3 activation and the molecular mechanisms involved remain incompletely understood. EXPERIMENTAL APPROACH: The expression of HSP90 and the activation of STAT3 were detected in muscle from the patients with cancer cachexia or the tumour-bearing cachectic mice. HSP90 inhibitors, including 17DMAG (alvespimycin) and PU-H71, were administered to cachexic mice and cachexia parameters, weight loss, food intake, survival rate, body composition, serum metabolites, muscle wasting pathology and catabolic activation were analysed. The co-culture of C2C12 myotube cells with C26 conditioned media was performed to investigate the pathological mechanism involved in catabolic muscle wasting. The roles of HSP90, STAT3 and FOXO1 in myotube atrophy were explored via overexpression or knockdown. RESULTS: An enhanced interaction between activated STAT3 and HSP90 in the skeletal muscle of cancer cachexia patients, is a crucial for the development of cachectic muscle wasting. HSP90 inhibitors 17DMAG and PU-H71 alleviated the muscle wasting in C26 and models or the myotube atrophy of C2C12 cells induced by C26 conditional medium. Prolonged STAT3 activation transactivated FOXO1 by binding directly to its promoter and triggered the muscle wasting in a FOXO1-dependent manner in muscle cells. CONCLUSION AND IMPLICATIONS: The HSP90/STAT3/FOXO1 axis plays a critical role in cachectic muscle wasting, which might be a potential therapeutic target for the treatment of cancer cachexia.

An integrative transcriptome study reveals Ddit4/Redd1 as a key regulator of cancer cachexia in rodent models.

Cancer cachexia is a multifactorial metabolic syndrome that causes up to 20% of cancer-related deaths. Muscle atrophy, the hallmark of cancer cachexia, strongly impairs the quality of life of cancer patients; however, the underlying pathological process is still poorly understood. Investigation of the disease pathogenesis largely relies on cachectic mouse models. In our study, the transcriptome of the cachectic gastrocnemius muscle in the C26 xenograft model was integrated and compared with that of 5 more different datasets. The bioinformatic analysis revealed pivotal gene ontology (GO) terms and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways of the disease, and the key genes were validated. Construction of the protein-protein interaction network and the comparison of pathways enriched in cancer cachexia with 5 other muscle atrophy models revealed Ddit4 (DNA damage-inducible transcript 4), as a key protein in cancer cachexia. The higher expression of Ddit4 in cachectic muscle was further validated in animal models and cachectic cancer patients. Further study revealed that p38 induced the expression of Ddit4, which in turn inhibited the mTOR pathway in atrophic cells.

Quantitative proteomics reveals arsenic attenuates stem-loop binding protein stability via a chaperone complex containing heat shock proteins and ERp44.

Arsenic pollution impacts health of millions of people in the world. Inorganic arsenic is a carcinogenic agent in skin and lung cancers. The stem-loop binding protein (SLBP) binds to the stem-loop of the canonical histone mRNA and regulates its metabolism during cell cycle. Our previous work has shown arsenic induces ubiquitin-proteasome dependent degradation of SLBP and contributes to lung cancer. In this study, we established the first comprehensive SLBP interaction network by affinity purification-mass spectrometry (AP-MS) analysis, and further demonstrated arsenic enhanced the association between SLBP and a crucial chaperone complex containing heat shock proteins (HSPs) and ERp44. Strikingly, knockdown of these proteins markedly rescued the protein level of SLBP under arsenic exposure conditions, and abolished the increasing migration capacity of BEAS-2B cells induced by arsenic. Taken together, our study provides a potential new mechanism that a chaperone complex containing HSPs and ERp44 attenuates the stability of SLBP under both normal and arsenic exposure conditions, which could be essential for arsenic-induced high cell migration.

Inhibiting HDAC3 (Histone Deacetylase 3) Aberration and the Resultant Nrf2 (Nuclear Factor Erythroid-Derived 2-Related Factor-2) Repression Mitigates Pulmonary Fibrosis.

[Figure: see text].

Soluble IL-6R-mediated IL-6 trans-signaling activation contributes to the pathological development of psoriasis.

IL-6 has been suggested to function as an autocrine mitogen in the psoriatic epidermis. The biological activity of IL-6 relies on interactions with its receptors, including the membrane-bound IL-6 receptor (mIL-6R) and soluble IL-6 receptor (sIL-6R). Our study presents data showing that the levels of plasma IL-6 and sIL-6R were elevated in psoriatic patients. Genotyping of two single-nucleotide polymorphisms (SNPs) in IL-6R (rs4845617 and rs2228145) demonstrated that the SNP IL-6R (rs4845617) rather than IL-6R (rs2228145) shows a significant association with psoriasis (P = 0.006). To verify the functions of sIL-6R, cultured keratinocytes and imiquimod (IMQ)-induced psoriatic model mice were treated with sIL-6R. We found that the presence of sIL-6R in the HaCaT cell culture medium enhanced the IL-6-induced Stat3 activation, which resulted in abnormal keratinocyte proliferation and aberrant differentiation. Furthermore, the application of sIL-6R in vivo accelerated the pathological development of the disease. Our results demonstrate for the first time that genetic polymorphisms in the IL-6R gene are associated with psoriasis disease phenotypes in a Chinese psoriatic patient population; sIL-6R-mediated trans-signaling pathway plays a pivotal role in keratinocyte proliferation and differentiation, suggesting potential therapeutics for psoriasis. KEY MESSAGES: Patients with psoriasis displayed higher levels of IL-6 and sIL-6R compared with healthy controls. Analysis of genotypes revealed that IL-6R rs4845617 GG genotype associated with the risk of psoriasis. Supplement of sIL-6R further enhanced IL-6-induced Stat3 activation in keratinocytes. In vivo administration of sIL-6R accelerated, whereas sgp130FC alleviated, the pathological development of psoriasis.

Genetic etiology study of four Chinese families with two nonsyndromic deaf children in succession by targeted next-generation sequencing.

BACKGROUND: Genetic components contribute significantly to the cause of hearing loss. Nonsyndromic hearing loss has been shown to have high genetic heterogeneity. For families who had given birth to two nonsyndromic deaf children in succession, it seems that their deafness was highly related to genetics. OBJECTIVES: This study aimed to disclose the genetic causes of the subjects from the four Chinese families with two nonsyndromic deaf children in succession who failed to find the genetic etiology of the hearing loss by common deafness genetic screening (GJB2, GJB3, SLC26A4, and MT-RNR1, including 20 hot variants in 4 genes). METHODS: Targeted next-generation sequencing (NGS) of 127 known deafness genes was performed in probands of four families, followed by a series of comprehensive analyses of all family members combined with a literature review of related genes. RESULTS: We identified pathogenic variants in three families including c.919-2A>G/c.1985G>A in SLC26A4; c.109G>A (p.V37I) in GJB2; and m.7505T>C in MT-TS1. Sanger sequencing confirmed that these variants segregated with the hearing impairment of each family. We also identified c.331C>T/c.625-5C>T/c.5717G>A in CDH23; c.138T>C in POU3F4 in two families, in which the pathogenicity in clinical was likely pathogenic or unknown. CONCLUSIONS: Using the NGS detection technology, we found the genetic etiology of the HL in part of deaf families. Our study provided a useful piece of information for the variant spectrum of hearing loss in Chinese families with two deaf children in succession.

Respiratory exposure to PM2.5 soluble extract disrupts mucosal barrier function and promotes the development of experimental asthma.

BACKGROUND: Air pollutants are important factors that contribute to the development and exacerbation of asthma, but experimental evidence still needs to be collected and the mechanisms still need to be addressed. In this study, we aimed to assess the association between PM2.5 exposure and asthma development. The effects of PM2.5 exposure on the barrier functions of airway epithelial cells were also determined. METHODS: PM2.5 was collected from Nanjing, China, and its soluble extract was prepared. Human lung epithelial cells (BEAS-2B) were treated with different concentrations of soluble PM2.5 extract, and cell viability was detected by FACS using Annexin V-FITC staining. PM2.5-induced oxidative stress and inflammatory events were assessed by DCF-DA staining and qPCR. PM2.5-induced dysfunction of the airway epithelial barrier was assessed by measuring the expression of tight junction molecules. In vivo, BALB/c mice were treated with OVA in the presence or absence of PM2.5 solution, followed by exposure to OVA aerosols. Allergy-induced airway inflammation and lung injury were assessed by histopathological analyses. RESULTS: Soluble PM2.5 extract exposure in vitro decreased the viability and increased apoptosis of airway epithelial cells. Soluble PM2.5 extract induced oxidative stress and enhanced pro-inflammatory factor expression by activating the NF-κB and MAPK signalling pathways, which were accompanied by reduced airway barrier function. The in vivo data demonstrated that PM2.5 exposure increased the effects of allergy sensitization after respiratory exposure to allergens, which led to the development of asthma. CONCLUSION: This study suggests that exposure to soluble PM2.5 extract contributes to airway barrier dysfunction. The soluble mediators generated by airway epithelial cells in response to PM2.5 exposure orchestrate the breaking of inhalational tolerance and sensitization to allergic antigens, leading to the exacerbated development of asthma.

FLCN-regulated miRNAs suppressed reparative response in cells and pulmonary lesions of Birt-Hogg-Dubé syndrome.

BACKGROUND: Birt-Hogg-Dubé Syndrome (BHDS) characterised by skin fibrofolliculomas, kidney tumour and pulmonary cysts/pneumothorax is caused by folliculin (FLCN) germline mutations. The pathology of both neoplasia and focused tissue loss of BHDS strongly features tissue-specific behaviour of the gene. Isolated cysts/pneumothorax is the most frequent atypical presentation of BHDS and often misdiagnosed as primary spontaneous pneumothorax (PSP). Deferential diagnosis of BHDS with isolated pulmonary presentation (PSP-BHD) from PSP is essential in lifelong surveillance for developing renal cell carcinoma. METHODS: The expression profiles of microRNAs (miRNAs) in cystic lesions of PSP-BHD and PSP were determined via microarray. The selected upregulated miRNAs were further confirmed in the plasma of an expanded cohort of PSP-BHD patients by reverse transcription quantitative PCR (RT-qPCR). Their diagnostic accuracy was evaluated. Moreover, the cellular functions and targeted signalling pathways of FLCN-regulated miRNAs were assessed in various cell lines and in the lesion tissue contexts. RESULTS: Cystic lesions of PSP-BHD and PSP showed different miRNAs profiles with a significant upregulation of miR-424-5p and let-7d-5p in PSP-BHD. The combination of the two effectively predicted BHDS patients. In vitro studies revealed a suppressive effect of FLCN on miR-424-5p and let-7d-5p expressions specifically in lung epithelial cells. The ectopic miRNAs triggered epithelial apoptosis and epithelial transition of mesenchymal cells and suppressed the reparative responses in cells and tissues with FLCN deficiency. CONCLUSION: The upregulation of miR-424-5p and let-7d-5p by FLCN deficiency occurred in epithelial cells and marked the PSP-BHD condition, which contributed to a focused degenerative pathology in the lung of PSP-BHD patients.

IKK-Mediated Regulation of the COP9 Signalosome via Phosphorylation of CSN5.

The COP9 signalosome (CSN) is an evolutionarily conserved multisubunit protein complex, which controls protein degradation through deneddylation and inactivation of cullin-RING ubiquitin E3 ligases (CRLs). Recently, the CSN complex has been linked to the NF-κB signaling pathway due to its association with the IKK complex. However, how the CSN complex is regulated in this signaling pathway remains unclear. Here, we have carried out biochemical experiments and confirmed the interaction between the CSN and IKK complexes. In addition, we have determined that overexpression of IKKα or IKKß leads to enhanced phosphorylation of CSN5, the catalytic subunit for CSN deneddylase activity. Mutational analyses have revealed that phosphorylation at serine 201 and threonine 205 of CSN5 impairs CSN-mediated deneddylation activity in vitro. Interestingly, TNF-α treatment not only enhances the interaction between CSN and IKK but also induces an IKK-dependent phosphorylation of CSN5 at serine 201, linking CSN to TNF-α signaling through IKK. Moreover, TNF-α treatment affects the CSN interaction network globally, especially the associations of CSN with the proteasome complex, eukaryotic translation initiation factor complex, and CRL components. Collectively, our results provide new insights into IKK-mediated regulation of CSN associated with the NF-κB signaling pathway.

A rapid NGS strategy for comprehensive molecular diagnosis of Birt-Hogg-Dubé syndrome in patients with primary spontaneous pneumothorax.

BACKGROUND: Primary spontaneous pneumothorax (PSP) or pulmonary cysts is one of the manifestations of Birt-Hogg-Dube syndrome (BHDS) that is caused by heterozygous mutations in FLCN gene. Most of the mutations are SNVs and small indels, and there are also approximately 10 % large intragenic deletions and duplications of the mutations. These molecular findings are generally obtained by disparate methods including Sanger sequencing and Multiple Ligation-dependent Probe Amplification in the clinical laboratory. In addition, as a genetically heterogeneous disorder, PSP may be caused by mutations in multiple genes include FBN1, COL3A1, CBS, SERPINA1 and TSC1/TSC2 genes. For differential diagnosis, these genes should also be screened which makes the diagnostic procedure more time-consuming and labor-intensive. METHODS: Forty PSP patients were divided into 2 groups. Nineteen patients with different pathogenic mutations of FLCN previously identified by conventional Sanger sequencing and MLPA were included in test group, 21 random PSP patients without any genetic screening were included in blinded sample group. 7 PSP genes including FLCN, FBN1, COL3A1, CBS, SERPINA1 and TSC1/TSC2 were designed and enriched by Haloplex system, sequenced on a Miseq platform and analyzed in the 40 patients to evaluate the performance of the targeted-NGS method. RESULTS: We demonstrated that the full spectrum of genes associated with pneumothorax including FLCN gene mutations can be identified simultaneously in multiplexed sequence data. Noteworthy, by our in-house copy number analysis of the sequence data, we could not only detect intragenic deletions, but also determine approximate deletion junctions simultaneously. CONCLUSIONS: NGS based Haloplex target enrichment technology is proved to be a rapid and cost-effective screening strategy for the comprehensive molecular diagnosis of BHDS in PSP patients, as it can replace Sanger sequencing and MLPA by simultaneously detecting exonic and intronic SNVs, small indels, large intragenic deletions and determining deletion junctions in PSP-related genes.

Promoter methylation is not associated with FLCN irregulation in lung cyst lesions of primary spontaneous pneumothorax.

Germline mutations in FLCN are responsible for ~10% of patients with primary spontaneous pneumothorax (PSP), characterized by multiple lung cysts in the middle/lower lobes and recurrent pneumothorax. These clinical features are also observed in a substantial portion of patients with sporadic PSP exhibiting no FLCN coding mutations. To assess the potential underlying mechanisms, 71 patients with PSP were selected, including 69 sporadic and 2 familial cases, who bared FLCN mutation­like lung cysts, however, harbored no FLCN protein­altering mutations. Notably, in a significant proportion of the patients, FLCN irregulation was observed at the transcript and protein levels. Genetic analyses of the cis­regulatory region of FLCN were performed by sequencing and multiplex ligation­dependent probe amplification assay. No inheritable DNA defect was detected, with the exception of a heterozygous deletion spanning the FLCN promoter, which was identified in a family with PSP. This mutation caused a reduction in the expression of FLCN in the lung cysts. Pedigree analysis demonstrated that haploinsufficiency of FLCN was pathogenic. To determine whether epigenetic mechanisms may be involved in the irregulation of FLCN, the promoter methylation status was measured in the remainder of the patients. No evidence of FLCN promoter methylation was demonstrated. The present study suggested that FLCN irregulation in lung cysts of PSP is not associated with promoter methylation.

Does familial breast cancer and thymoma suggest a cancer syndrome? A family perspective.

Concurrence of breast cancer or thymoma with other malignancies in individual families is often observed, but the familial concurrence of breast cancer and thymoma has not yet been reported. Herein we reported a family encompassing five breast/ovarian cancer patients and two thymoma patients. Whole genome linkage analysis detected no haplotype co-segregating with both types of the tumors. In all patients with breast/ovarian cancer, genetic analysis revealed a clinically untested variant c.5141T>G in exon 18 of the BRCA1 gene, which could be a cancer-causing variant based on the functional study of Lee et al. (2010) and our current pedigree analysis. In the two thymoma patients in our family, targeted sequencing of RAD51L1 and BMP2 genes in and near the translocation site of chromosome 14 and 20 previously reported in two thymoma families, did not find any pathogenic mutation. In the present study, we identified a clinically unconfirmed BRCA1 variant segregating with breast/ovarian cancer patients in an individual family, suggesting it to be clinically functional. Our evidence, however, did not support the notion that the concurrent appearance of breast cancer and thymoma in our family represents a familial cancer syndrome caused by the same genetic disorder.

FLCN intragenic deletions in Chinese familial primary spontaneous pneumothorax.

Primary spontaneous pneumothorax (PSP) is a significant clinical problem, affecting tens of thousands patients annually. Germline mutations in the FLCN gene have been implicated in etiology of familial PSP (FPSP). Most of the currently identified FLCN mutations are small indels or point mutations that detected by Sanger sequencing. The aim of this study was to determine large FLCN deletions in PSP families that having no FLCN sequence-mutations. Multiplex ligation-dependent probe amplification (MLPA) assays and breakpoint analyses were used to detect and characterize the deletions. Three heterozygous FLCN intragenic deletions were identified in nine unrelated Chinese families including the exons 1-3 deletion in two families, the exons 9-14 deletion in five families and the exon 14 deletion in two families. All deletion breakpoints are located in Alu repeats. A 5.5 Mb disease haplotype shared in the five families with exons 9-14 deletion may date the appearance of this deletion back to approximately 16 generations ago. Evidences for founder effects of the other two deletions were also observed. This report documents the first identification of founder mutations in FLCN, as well as expands mutation spectrum of the gene. Our findings strengthen the view that MLPA analysis for intragenic deletions/duplications, as an important genetic testing complementary to DNA sequencing, should be used for clinical molecular diagnosis in FPSP.