H3K4me3 as a target of di(2-ethylhexyl) phthalate (DEHP) impairing primordial follicle assembly.

Di (2-ethylhexyl) phthalate (DEHP) is a widely used plastics additive that growing evidence indicates as endocrine disruptor able to negatively affect various reproductive processes both in female and male animals, including humans. However, the precise molecular mechanism of such actions is not completely understood. In the present study, scRNA-seq was performed on the ovaries of offspring from mothers exposed to DEHP from 16.5 days post coitum to 3 days post-partum, when the primordial follicle (PF) stockpile is established. While the histological observations of the offspring ovaries from DEHP exposed mothers confirmed previous data about a distinct reduction of oocytes enclosed in PFs. Focusing on oocytes, scRNA-seq analyses showed that the genes that mostly changed by DEHP were enriched GO terms related to histone H3-K4 methylation. Moreover, we observed H3K4me3 level, an epigenetics modification of H3 that is crucial for chromatin transcription, decreased by 40.28% (P < 0.01) in DEHP-treated group compared with control. When the newborn ovaries were cultured in vitro, the DEHP effects were abolished by tamoxifen (an estrogen receptor antagonist) or overexpression of Smyd3 (one specific methyltransferase of H3K4me3), in particular, the percentage of oocyte enclosed in PF was increased by 15.39% in DEHP plus Smyd3 overexpression group than of DEHP group (P < 0.01), which was accompanied by the upregulation of H3K4me3. Collectively, the present results discover Smyd3-H3K4me3 as a novel target of the deleterious ER-mediated effect of DEHP on PF formation during early folliculogenesis in the mouse and highlight epigenetics changes as prominent targets of endocrine disruptors like DEHP.

Jianpi Qinghua Fomula alleviates insulin resistance via restraining of MAPK pathway to suppress inflammation of the small intestine in DIO mice.

BACKGROUND: Jianpi Qinghua Fomula (JPQHF), a clinically proven prescription,has been applied to cure insulin resistance(IR) and type 2 diabetes (T2DM) for more than 20 years. Here, we will unravel the underlying molecular mechanisms relevant to the therapeutic actions of JPQHF. METHODS: High-fat(HF)diet-induced obesity(DIO)mouse were established in our research, along with insulin resistance. After the administration of JPQHF 5 or 6 weeks, the parameters of the glucose and lipid metabolism were measured. Flow cytometry and Luminex were utilized to assess the inflammation in small intestine,whilst Western blot was used to determine the relative expression levels of the MAPK pathway-related proteins. The glucose and lipid transporter of small intestine was assessed by immunofluorescence and ELISA, and the expression of insulin signaling pathway was detected by Western blot. RESULTS: The metabolic phenotypes of DIO mouse were ameliorated after 6-week oral administration of JPQHF; Meanwhile,JPQHF downregulated levels of IL-1ß,IL-6, TNF-α and IFN-γ but upregulated the ratio of M2/M1 macrophages in the small intestine. The elevated expressions of p-P38 MAPK/P38 MAPK、p-JNK/JNK and p-ERK1/2/ERK1/2 were reversed by JPQHF. Moreover, JPQHF enhanced expression of PI3K,p-AKT/AKT, p-IRS1/ IRS1, p-IRS2/ IRS2 and apoB48 in small intestine, and facilitated the translocation of GLUT2 to the basal side of small intestine epithelial cells. CONCLUSION: JPQHF alleviates insulin resistance in DIO mice, and this effect may be associated with its restraining of inflammation of small intestine via attenuating MAPK pathway, and then diminishes small intestinal glucose and lipid absorption.

Oxidative stress as a plausible mechanism for zearalenone to induce genome toxicity.

Zearalenone (ZEN), a common non-steroidal estrogenic mycotoxin of the Fusarium genus, is one of the most frequent and powerful contaminant of grains and cereal products representing a serious threat for people and livestock health. In fact, ZEN causes cytotoxicity and genotoxicity in a variety of cell types at least in part through binding to estrogen receptors (ERs). The main pathways through which ZEN induces such effects remain, however, elusive. In particular, how the mycotoxin causes DNA damage, dysregulates DNA repair mechanisms, changes epigenome of targeted cells and, not least, affects chromatin conformation and non-coding RNA (ncRNA), is unclear. In the present paper, following extensive review of the literature about such ZEN effects and our own experience in studying the effects of this compound on reproductive processes, we propose that increased production of reactive oxygen species (ROS) and consequently oxidative stress (OS) are central in ZEN genotoxicity. Besides to shed light on the action mechanisms of the mycotoxin, this notion might help to develop effective strategies to counteract its deleterious biological effects.

Genetic Regulation of N6-Methyladenosine-RNA in Mammalian Gametogenesis and Embryonic Development.

Emerging evidence shows that m6A is the most abundant modification in eukaryotic RNA molecules. It has only recently been found that this epigenetic modification plays an important role in many physiological and pathological processes, such as cell fate commitment, immune response, obesity, tumorigenesis, and relevant for the present review, gametogenesis. Notably the RNA metabolism process mediated by m6A is controlled and regulated by a series of proteins termed writers, readers and erasers that are highly expressed in germ cells and somatic cells of gonads. Here, we review and discuss the expression and the functional emerging roles of m6A in gametogenesis and early embryogenesis of mammals. Besides updated references about such new topics, readers might find in the present work inspiration and clues to elucidate epigenetic molecular mechanisms of reproductive dysfunction and perspectives for future research.

DrABC: deep learning accurately predicts germline pathogenic mutation status in breast cancer patients based on phenotype data.

BACKGROUND: Identifying breast cancer patients with DNA repair pathway-related germline pathogenic variants (GPVs) is important for effectively employing systemic treatment strategies and risk-reducing interventions. However, current criteria and risk prediction models for prioritizing genetic testing among breast cancer patients do not meet the demands of clinical practice due to insufficient accuracy. METHODS: The study population comprised 3041 breast cancer patients enrolled from seven hospitals between October 2017 and 11 August 2019, who underwent germline genetic testing of 50 cancer predisposition genes (CPGs). Associations among GPVs in different CPGs and endophenotypes were evaluated using a case-control analysis. A phenotype-based GPV risk prediction model named DNA-repair Associated Breast Cancer (DrABC) was developed based on hierarchical neural network architecture and validated in an independent multicenter cohort. The predictive performance of DrABC was compared with currently used models including BRCAPRO, BOADICEA, Myriad, PENN II, and the NCCN criteria. RESULTS: In total, 332 (11.3%) patients harbored GPVs in CPGs, including 134 (4.6%) in BRCA2, 131 (4.5%) in BRCA1, 33 (1.1%) in PALB2, and 37 (1.3%) in other CPGs. GPVs in CPGs were associated with distinct endophenotypes including the age at diagnosis, cancer history, family cancer history, and pathological characteristics. We developed a DrABC model to predict the risk of GPV carrier status in BRCA1/2 and other important CPGs. In predicting GPVs in BRCA1/2, the performance of DrABC (AUC = 0.79 [95% CI, 0.74-0.85], sensitivity = 82.1%, specificity = 63.1% in the independent validation cohort) was better than that of previous models (AUC range = 0.57-0.70). In predicting GPVs in any CPG, DrABC (AUC = 0.74 [95% CI, 0.69-0.79], sensitivity = 83.8%, specificity = 51.3% in the independent validation cohort) was also superior to previous models in their current versions (AUC range = 0.55-0.65). After training these previous models with the Chinese-specific dataset, DrABC still outperformed all other methods except for BOADICEA, which was the only previous model with the inclusion of pathological features. The DrABC model also showed higher sensitivity and specificity than the NCCN criteria in the multi-center validation cohort (83.8% and 51.3% vs. 78.8% and 31.2%, respectively, in predicting GPVs in any CPG). The DrABC model implementation is available online at http://gifts.bio-data.cn/ . CONCLUSIONS: By considering the distinct endophenotypes associated with different CPGs in breast cancer patients, a phenotype-driven prediction model based on hierarchical neural network architecture was created for identification of hereditary breast cancer. The model achieved superior performance in identifying GPV carriers among Chinese breast cancer patients.

Maternal Zearalenone exposure impacted ovarian follicle formation and development of suckled offspring.

Zearalenone (ZEN) is a secondary metabolite, which is mainly produced by Fusarium fungi and exists in various feeds and agricultural products. Recently, an increasing amount of data has shown that ZEN, as an estrogen-like hormone, can have harmful effects on the female reproductive system, especially on oogenesis and folliculogenesis. Breast milk is considered to be the ideal form of nutrition for infants; however, there are some records of contaminants in food, such as mycotoxins, which may be transferred from maternal blood to milk. In this study, we investigated the toxic effects of breast milk on folliculogenesis in offspring following maternal ZEN exposure. Our results showed that maternal ZEN exposure significantly inhibited the process of primordial follicle (PF) assembly and reduced the number of PFs in suckled offspring's ovaries. In addition, RNA-seq analysis showed that RIG-I-like receptor (RLRs) signaling pathways were activated after exposed to ZEN, which increased the expression levels of DNA damage (γ-H2AX, RAD51, and PARP1) and apoptosis related protein (BAX/BCL2 and Caspase-3). Finally, ZEN exposure interfered with follicular development, as evidenced by the reduced percentages of oocyte maturation and embryonic development when the offspring grew to adolescence. It is worth noting that maternal ZEN exposure disrupted the tri-methylation levels of H3K4, H3K9, and H3K27 in the offspring's oocytes. Our results indicated that maternal ZEN exposure affected ovarian development in offspring through the breast milk, which may be detrimental to their reproductive capability in adult life.

Deciphering the mutational signature of congenital limb malformations.

Congenital limb malformations (CLMs) affect 1 in 500 live births. However, the value of exome sequencing (ES) for CLM is lacking. The purpose of this study was to decipher the mutational signature of CLM on an exome level. We enrolled a cohort of 66 unrelated probands (including 47 families) with CLM requiring surgical correction. ES was performed for all patients and available parental samples. A definite molecular diagnosis was achieved in 21 out of 66 (32%) patients. We identified 19 pathogenic or likely pathogenic single-nucleotide variants and three copy number variants, of which 11 variants were novel. We identified four variants of uncertain significance. Additionally, we identified RPL9 and UBA2 as novel candidate genes for CLM. By comparing the detailed phenotypic features, we expand the phenotypic spectrum of diastrophic dysplasia and chromosome 6q terminal deletion syndrome. We also found that the diagnostic rate was significantly higher in patients with a family history of CLM (p = 0.012) or more than one limb affected (p = 0.034). Our study expands our understanding of the mutational and phenotypic spectrum of CLM and provides novel insights into the genetic basis of these syndromes.

Inflammatory cytokines as key players of apoptosis induced by environmental estrogens in the ovary.

Natural and synthetic environmental estrogens (EEs), interfering with the physiological functions of the body's estrogens, are widespread and are rising much concern for their possible deleterious effects on human and animal health, in particular on reproduction. In fact, increasing evidence indicate that EEs can be responsible for a variety of disfunctions of the reproductive system especially in females such as premature ovarian insufficiency (POI). Because of their great structural diversity, the modes of action of EEs are controversial. One important way through which EEs exert their effects on reproduction is the induction of apoptosis in the ovary. In general, EEs can exert pro-and anti-apoptotic effects by agonizing or antagonizing numerous estrogen-dependent signaling pathways. In the present work, results concerning apoptotic pathways and diseases induced by representative EEs (such as zearalenone, bisphenol A and di-2-ethylhexyl phthalate), in ovaries throughout development are presented into an integrated network. By reviewing and elaborating these studies, we propose inflammatory factors, centered on the production of tumor necrosis factor (TNF), as a major cause of the induction of apoptosis by EEs in the mammalian ovary. As a consequence, potential strategies to prevent such EE effect are suggested.

Variants Affecting the C-Terminal of CSF1R Cause Congenital Vertebral Malformation Through a Gain-of-Function Mechanism.

CSF1R encodes the colony-stimulating factor 1 receptor which regulates the proliferation, differentiation, and biological activity of monocyte/macrophage lineages. Pathogenic variants in CSF1R could lead to autosomal dominant adult-onset leukoencephalopathy with axonal spheroids and pigmented glia or autosomal recessive skeletal dysplasia. In this study, we identified three heterozygous deleterious rare variants in CSF1R from a congenital vertebral malformation (CVM) cohort. All of the three variants are located within the carboxy-terminal region of CSF1R protein and could lead to an increased stability of the protein. Therefore, we established a zebrafish model overexpressing CSF1R. The zebrafish model exhibits CVM phenotypes such as hemivertebral and vertebral fusion. Furthermore, overexpression of the mutated CSF1R mRNA depleted of the carboxy-terminus led to a higher proportion of zebrafish with vertebral malformations than wild-type CSF1R mRNA did (p = 0.03452), implicating a gain-of-function effect of the C-terminal variant. In conclusion, variants affecting the C-terminal of CSF1R could cause CVM though a potential gain-of-function mechanism.

Phenotypic and genetic spectrum of isolated macrodactyly: somatic mosaicism of PIK3CA and AKT1 oncogenic variants.

BACKGROUND: Isolated macrodactyly is a severe congenital hand anomaly with functional and physiological impact. Known causative genes include PIK3CA, AKT1 and PTEN. The aim of this study is to gain insights into the genetics basis of isolated macrodactyly. RESULTS: We enrolled 24 patients with isolated macrodactyly. Four of them were diagnosed with Proteus syndrome based on skin presentations characteristic to this disease. Targeted next-generation sequencing was performed using patients' blood and affected tissues. Overall, 20 patients carry mosaic PIK3CA pathogenic variants, i.e. p.His1047Arg (N = 7), p.Glu542Lys (N = 6), p.Glu545Lys (N = 2), p.His1047Leu (N = 2), p.Glu453Lys (N = 1), p.Gln546Lys (N = 1) and p.His1047Tyr (N = 1). Four patients who met the diagnostic criteria of Proteus syndrome carry mosaic AKT1 p.Glu17Lys variant. Variant allele frequencies of these mosaic variants obtained through next-generation sequencing range from 10 to 33%. In genotype-phenotype correlation analysis of patients with PIK3CA variant, we found that patients with the macrodactyly of the lower limbs tend to carry PIK3CA variants located in the helical domain (P = 0.005). CONCLUSIONS: Mosaic PIK3CA and AKT1 variants can be found in all of our samples with isolated macrodactyly. Insights into phenotypic and genetic spectrum of isolated macrodactyly may be helpful in perusing a more precise and effective management of isolated macrodactyly.

Maternal Bisphenol S exposure affects the reproductive capacity of F1 and F2 offspring in mice.

Bisphenol S (BPS) is an endocrine disruptor which is widely used in commercial plastic products. Previous studies have shown that exposure to BPS has toxic effects on various aspects of mammalian, but there are few reports about reproductive toxicity. In order to investigate the effects of maternal BPS exposure on the reproductive of F1 and F2 female mice, the pregnant mice were orally administered with different dosages of BPS only once every day from 12.5 to 15.5 days post-coitus (dpc). The results showed that maternal BPS exposure to 2 µg per kg of body weight per day (2 µg/kg) and 10 µg/kg accelerated the meiotic prophase I (MPI) of F1 female mice and the expression of the genes related to meiotic were increased. Further studies showed that maternal BPS exposure resulted in a significant increase in the percentage of oocytes enclosed in primordial follicles in the 3 days post-partum (3 dpp) ovaries of F1 female mice. And at the time of 21 days post-partum (21 dpp) in F1 female mice, the number of antral follicles were significantly lower compare to controls. In the study of five-week female mice of F1, we found that BPS disturbed the folliculogenesis, and the maturation rates and fertilization rates of oocytes were significantly decreased. Of note, maternal BPS exposure disrupted H3K4 and H3K9 tri-methylation levels in F1 ovaries. Maternal BPS exposure only affected the cyst breakdown in F2 female mice. Taken together, our results suggest that, maternal BPS exposure impaired the process of meiosis and oogenesis of F1 and F2 offspring, resulting in abnormal follicular development and serious damage to the reproduction.

Effects of Ochratoxin A exposure on DNA damage in porcine granulosa cells in vitro.

Ochratoxin A (OTA), a feed mycotoxin, tends to impair the reproductive performance of animals. Our previous studies have demonstrated that OTA exposure inhibits porcine ovarian granulosa cell (GC) proliferation and induces their apoptosis, but the underlying toxic mechanism is still uncertain. In this study, we explored the OTA exposure on porcine GCs in vitro and found that OTA exposure inhibited the proliferation of porcine GCs and arrested cell cycle of GCs in the G2/M phase. The results based on RNA-Seq revealed that 20 µM and 40 µM OTA exposure increase DNA damage of porcine GCs in vitro. The differentially expressed genes (DEGs) of 40 µM OTA exposure were enriched in the pathways of mismatch repair, nucleotide excision repair and homologous recombination in DNA replication compared with control group and 20 µM OTA exposure group. Meanwhile, OTA exposure increased the expression levels of DNA double-strand breaks (DSBs) gene γ-H2AX, and DNA repair related genes, such as BRCA1, XRCC1, PARP1, and RAD51. Above all, our research revealed that OTA might exert deleterious effects on porcine ovarian GCs, influencing DNA repair-related biological processes and causing DNA damage response.

A novel COMP mutation in a Chinese family with multiple epiphyseal dysplasia.

BACKGROUND: Multiple epiphyseal dysplasia (MED) is a skeletal disorder characterized by delayed and irregular ossification of the epiphyses and early-onset osteoarthritis. At least 66% of the reported autosomal dominant MED (AD-MED) cases are caused by COMP mutations. METHODS: We recruited a four-generation Chinese family with early-onset hip osteoarthritis, flatfoot, brachydactyly, and mild short stature. An assessment of the family history, detailed physical examinations, and radiographic evaluations were performed on the proband and other family members, followed by the performance of whole-exome sequencing (WES). The pathogenicity of the candidate mutation was also analyzed. RESULTS: An AD-MED family with 10 affected members and 17 unaffected members was recruited. The main radiographic findings were symmetrical changes in the dysplastic acetabulum and femoral heads, irregular contours of the epiphyses, a shortened femoral neck, and flatfoot. Lower bone density was also observed in the ankle joints, wrist joints, and knees, as well as irregular vertebral end plates. In the proband, we identified the missense mutation c.1153G > T (p. Asp385Tyr), located in exon 11 of the COMP gene. This mutation was assessed as 'pathogenic' because of its low allele frequency and its high likelihood of co-segregation with disease in the reported family. Sanger sequencing validated the novel heterozygous mutation c.1153G > T (p. Asp385Tyr) in exon 11 of COMP in all affected individuals in the family. CONCLUSIONS: Our results underlined a key role of the Asp385 amino acid in the protein function of COMP and confirmed the pathogenicity of the COMP (c.1153G > T; p. Asp385Tyr) mutation in AD-MED disease. We have therefore expanded the known mutational spectrum of COMP and revealed new phenotypic information for AD-MED.

Estrogen Receptors (ESRs) Mutations in Adolescent Idiopathic Scoliosis: A Cross-Sectional Study.

BACKGROUND Adolescent idiopathic scoliosis (AIS) is the most common spinal deformity, but its etiology is unclear. Multiple genetic mutations have been reported to be associated with AIS. MATERIAL AND METHODS We enrolled a cohort of 113 surgically treated AIS patients with available parental subjects from the Peking Union Medical College Hospital. We performed whole-exome sequencing in 10 trio families and whole-genome sequencing in 103 singleton patients. Luciferase assay was used to detect the functional alterations of candidate ESR1 and ESR2 variants. RESULTS Using a de novo strategy, a missense variant in ESR1 (c.868A>G) was selected as a candidate gene for AIS. The main Cobb angle of this patient was 41° (T6-T10). Another potential pathogenic variant in ESR2 (c.236T>C) was identified. The main curve of the patient was 45° at T10-L3. The transactivation capacities of the mutated ESR1 and ESR2 protein were both significantly decreased (p=0.026 and 0.014, respectively). CONCLUSIONS Potential pathogenic variants in ESR1 and ESR2 were identified in 113 AIS patients, suggesting that genetic mutations in ESR1/2 were associated with the risk of AIS.

Exome sequencing reveals a novel variant in NFX1 causing intracranial aneurysm in a Chinese family.

BACKGROUND: Genetic risk factors play an important role in the pathogenesis of familial intracranial aneurysms (FIAs); however, the molecular mechanisms remain largely unknown. OBJECTIVE: To investigate potential FIA-causing genetic variants by rare variant interrogation and a family-based genomics approach in a large family with an extensive multigenerational pedigree with FIAs. METHOD: Exome sequencing (ES) was performed in a dominant likely family with intracranial aneurysms (IAs). Variants were analyzed by an in-house developed pipeline and prioritized using various filtering strategies, including population frequency, variant type, and predicted variant pathogenicity. Sanger sequencing was also performed to evaluate the segregation of the variants with the phenotype. RESULTS: Based on the ES data obtained from five individuals from a family with 7/21 living members affected with IAs, a total of 14 variants were prioritized as candidate variants. Familial segregation analysis revealed that NFX1 c.2519T>C (p.Leu840Pro) segregated in accordance with Mendelian expectations with the phenotype within the family-that is, present in all IA-affected cases and absent from all unaffected members of the second generation. This missense variant is absent from public databases (1000genome, ExAC, gnomAD, ESP5400), and has damaging predictions by bioinformatics tools (Gerp ++ score = 5.88, CADD score = 16.43, MutationTaster score = 1, LRT score = 0). In addition, 840Leu in NFX1 is robustly conserved in mammals and maps in a region before the RING-type zinc finger domain. CONCLUSION: NFX1 c.2519T>C (p.Leu840Pro) may contribute to the pathogenetics of a subset of FIAs.

A Recurrent Rare SOX9 Variant (M469V) is Associated with Congenital Vertebral Malformations.

OBJECTIVE: The genetic variations contributed to a substantial proportion of congenital vertebral malformations (CVM). SOX9 gene, a member of the SOX gene family, has been implicated in CVM. To study the SOX9 mutation in CVM patients is of great significance to explain the pathogenesis of scoliosis (the clinical manifestation of CVM) and to explore the pathogenesis of SOX9-related skeletal deformities. METHODS: A total of 50 singleton patients with CVM were included in this study. Exome Sequencing (ES) was performed on all the patients. The recurrent candidate variant of SOX9 gene was validated by Sanger sequencing. Luciferase assay was performed to investigate the functional changes of this variant. RESULTS: A recurrent rare heterozygous missense variant in SOX9 gene (NM_000346.3: c.1405A>G, p.M469V) which had not been reported previously was identified in three CVM patients who had the clinical findings of congenital scoliosis without deformities in other systems. This variant was absent from our in-house database and it was predicted to be deleterious (CADD = 24.5). The luciferase assay demonstrated that transactivation capacity of the mutated SOX9 protein was significantly lower than that of the wild-type for the two luciferase reporters (p = 0.0202, p = 0.0082, respectively). CONCLUSION: This SOX9 mutation (p.M469V) may contribute to CVM without other systematic deformity, which provides important implications and better understanding of phenotypic variability in SOX9-related skeletal deformities.

Whole-exome sequencing reveals known and novel variants in a cohort of intracranial vertebral-basilar artery dissection (IVAD).

Intracranial vertebral-basilar artery dissection (IVAD) is an arterial disorder leading to life-threatening consequences. Genetic factors are known to be causative to certain syndromic forms of IVAD. However, systematic study of the molecular basis of sporadic and isolated IVAD is lacking. To identify genetic variants contributing to the etiology of IVAD, we enrolled a cohort of 44 unrelated cases with a clinical diagnosis of isolated IVAD and performed whole-exome sequencing (WES) for all the participants; a trio exome sequencing approach was used when samples from both parents were available. Four previously reported disease-causing heterozygous variants (three in COL3A1 and one in FBN1) and seven novel heterozygous variants in IVAD-related genes were identified. In addition, six variants in novel IVAD genes including two de novo heterozygous nonsynonymous variants (each in VPS52 and CDK18), two stop-gain variants (each in MYH9 and LYL1), and two heterozygous biallelic variants in TNXB were considered to be possibly contributing to the phenotype, with unknown significance according to the existing knowledge. A significantly higher mutational rate of IVAD candidate genes was observed in patients versus our in-house controls (P = 0.002) (DISCO study, http://www.discostudy.org/ , n = 2248). Our study provided a mutational landscape for patients with isolated IVAD.

Perturbations of BMP/TGF-ß and VEGF/VEGFR signalling pathways in non-syndromic sporadic brain arteriovenous malformations (BAVM).

BACKGROUND: Brain arteriovenous malformations (BAVM) represent a congenital anomaly of the cerebral vessels with a prevalence of 10-18/100 000. BAVM is the leading aetiology of intracranial haemorrhage in children. Our objective was to identify gene variants potentially contributing to disease and to better define the molecular aetiology underlying non-syndromic sporadic BAVM. METHODS: We performed whole-exome trio sequencing of 100 unrelated families with a clinically uniform BAVM phenotype. Pathogenic variants were then studied in vivo using a transgenic zebrafish model. RESULTS: We identified four pathogenic heterozygous variants in four patients, including one in the established BAVM-related gene, ENG, and three damaging variants in novel candidate genes: PITPNM3, SARS and LEMD3, which we then functionally validated in zebrafish. In addition, eight likely pathogenic heterozygous variants (TIMP3, SCUBE2, MAP4K4, CDH2, IL17RD, PREX2, ZFYVE16 and EGFR) were identified in eight patients, and 16 patients carried one or more variants of uncertain significance. Potential oligogenic inheritance (MAP4K4 with ENG, RASA1 with TIMP3 and SCUBE2 with ENG) was identified in three patients. Regulation of sma- and mad-related proteins (SMADs) (involved in bone morphogenic protein (BMP)/transforming growth factor beta (TGF-ß) signalling) and vascular endothelial growth factor (VEGF)/vascular endotheliual growth factor recepter 2 (VEGFR2) binding and activity (affecting the VEGF signalling pathway) were the most significantly affected biological process involved in the pathogenesis of BAVM. CONCLUSIONS: Our study highlights the specific role of BMP/TGF-ß and VEGF/VEGFR signalling in the aetiology of BAVM and the efficiency of intensive parallel sequencing in the challenging context of genetically heterogeneous paradigm.

[A review of mixed gas detection system based on infrared spectroscopic technique].

In order to provide the experiences and references to the researchers who are working on infrared (IR) mixed gas detection field. The proposed manuscript reviews two sections of the aforementioned field, including optical multiplexing structure and detection method. At present, the coherent light sources whose representative are quantum cascade laser (QCL) and inter-band cascade laser(ICL) become the mainstream light source in IR mixed gas detection, which replace the traditional non-coherent light source, such as IR radiation source and IR light emitting diode. In addition, the photon detector which has a super high detectivity and very short response time is gradually beyond thermal infrared detector, dominant in the field of infrared detector. The optical multiplexing structure is the key factor of IR mixed gas detection system, which consists of single light source multi-plexing detection structure and multi light source multiplexing detection structure. Particularly, single light source multiplexing detection structure is advantages of small volume and high integration, which make it a plausible candidate for the portable mixed gas detection system; Meanwhile, multi light source multiplexing detection structure is embodiment of time division multiplex, frequency division multiplexing and wavelength division multiplexing, and become the leading structure of the mixed gas detection system because of its wider spectral range, higher spectral resolution, etc. The detection method applied to IR mixed gas detection includes non-dispersive infrared (NDIR) spectroscopy, wavelength and frequency-modulation spectroscopy, cavity-enhanced spectroscopy and photoacoustic spectroscopy, etc. The IR mixed gas detection system designed by researchers after recognizing the whole sections of the proposed system, which play a significant role in industrial and agricultural production, environmental monitoring, and life science, etc.