Genetic alterations in thyroid cancer mediating both resistance to BRAF inhibition and anaplastic transformation.

A subset of thyroid cancers present at advanced stage or with dedifferentiated histology and have limited response to standard therapy. Tumors harboring the BRAF V600E mutation may be treated with BRAF inhibitors; however, tumor response is often short lived due to multiple compensatory resistance mechanisms. One mode of resistance is the transition to an alternative cell state, which on rare occasions can correspond to tumor dedifferentiation. DNA sequencing and RNA expression profiling show that thyroid tumors that dedifferentiate after BRAF inhibition are enriched in known genetic alterations that mediate resistance to BRAF blockade, and may also drive tumor dedifferentiation, including mutations in the PI3K/AKT/MTOR (PIK3CA, MTOR), MAP/ERK (MET, NF2, NRAS, RASA1), SWI/SNF chromatin remodeling complex (ARID2, PBRM1), and JAK/STAT pathways (JAK1). Given these findings, recent investigations have evaluated the efficacy of dual-target therapies; however, continued lack of long-term tumor control illustrates the complex and multifactorial nature of these compensatory mechanisms. Transition to an immune-suppressed state is another correlate of BRAF inhibitor resistance and tumor dedifferentiation, suggesting a possible role for concurrent targeted therapy with immunotherapy. Investigations into combined targeted and immunotherapy are ongoing, but early results with checkpoint inhibitors, viral therapies, and CAR T-cells suggest enhanced anti-tumor immune activity with these combinations.

Lymphatic Malformations in Parkes Weber's Syndrome: Retrospective Review of 16 Cases in a Vascular Anomalies Center.

INTRODUCTION: Parkes Weber's syndrome (PWS) is a rare genetic disorder characterized by overgrowth and vascular malformations, primarily affecting the extremities. While PWS is known to be associated with arteriovenous and capillary malformations, the potential involvement of lymphatic malformations (LMs) has not been previously reported. The objective of this study is to investigate the presence of lymphatic anomalies in PWS patients and their role in the development of limb asymmetry. MATERIALS AND METHODS: This is a retrospective study of patients diagnosed with PWS in a Vascular Anomalies Center from 1994 to 2020. Clinical data were obtained from medical records including diagnostic imaging, lymphoscintigraphy, and genetic testing. The Institutional Review Board and Ethics Committee have approved this study. RESULTS: A total of 16 patients aged 18 interquartile range 14.7 years diagnosed with PWS were included (50% female). Six of the 16 patients with PWS had clinical and imaging data suggestive of LM (37.5%) and 3 of them had genetic variants in RASA1 (2/3) or KRAS (1/3). Limb asymmetry was greater in patients with isolated PWS (2.6 ± 0.8 cm) than in the PWS-lymphatic anomalies population (2 ± 0.7 cm), although not significant (p = 0.247). One in 6 patients with PWS-LM required amputation (16.6%) versus 1 in 10 in isolated PWS (10%). CONCLUSION: Lymphatic anomalies may be present in a significant number of patients with PWS and could have a role in limb asymmetry and outcomes. It is paramount to investigate their existence and distinguish them from true overgrowth.

Mutation of key signaling regulators of cerebrovascular development in vein of Galen malformations.

To elucidate the pathogenesis of vein of Galen malformations (VOGMs), the most common and most severe of congenital brain arteriovenous malformations, we performed an integrated analysis of 310 VOGM proband-family exomes and 336,326 human cerebrovasculature single-cell transcriptomes. We found the Ras suppressor p120 RasGAP (RASA1) harbored a genome-wide significant burden of loss-of-function de novo variants (2042.5-fold, p = 4.79 x 10-7). Rare, damaging transmitted variants were enriched in Ephrin receptor-B4 (EPHB4) (17.5-fold, p = 1.22 x 10-5), which cooperates with p120 RasGAP to regulate vascular development. Additional probands had damaging variants in ACVRL1, NOTCH1, ITGB1, and PTPN11. ACVRL1 variants were also identified in a multi-generational VOGM pedigree. Integrative genomic analysis defined developing endothelial cells as a likely spatio-temporal locus of VOGM pathophysiology. Mice expressing a VOGM-specific EPHB4 kinase-domain missense variant (Phe867Leu) exhibited disrupted developmental angiogenesis and impaired hierarchical development of arterial-capillary-venous networks, but only in the presence of a "second-hit" allele. These results illuminate human arterio-venous development and VOGM pathobiology and have implications for patients and their families.

rasa1-related arteriovenous malformation is driven by aberrant venous signalling.

Arteriovenous malformations (AVMs) develop where abnormal endothelial signalling allows direct connections between arteries and veins. Mutations in RASA1, a Ras GTPase activating protein, lead to AVMs in humans and, as we show, in zebrafish rasa1 mutants. rasa1 mutants develop cavernous AVMs that subsume part of the dorsal aorta and multiple veins in the caudal venous plexus (CVP) - a venous vascular bed. The AVMs progressively enlarge and fill with slow-flowing blood. We show that the AVM results in both higher minimum and maximum flow velocities, resulting in increased pulsatility in the aorta and decreased pulsatility in the vein. These hemodynamic changes correlate with reduced expression of the flow-responsive transcription factor klf2a. Remodelling of the CVP is impaired with an excess of intraluminal pillars, which is a sign of incomplete intussusceptive angiogenesis. Mechanistically, we show that the AVM arises from ectopic activation of MEK/ERK in the vein of rasa1 mutants, and that cell size is also increased in the vein. Blocking MEK/ERK signalling prevents AVM initiation in mutants. Alterations in venous MEK/ERK therefore drive the initiation of rasa1 AVMs.

Spectrum of lymphatic anomalies in patients with RASA1-related CM-AVM.

BACKGROUND: Capillary malformation-arteriovenous malformation (CM-AVM) is characterized by multifocal fast-flow capillary malformations, sometimes with arteriovenous malformations/fistulas, skeletal/soft tissue overgrowth, telangiectasias, or Bier spots. Lymphatic abnormalities are infrequently reported. We describe seven patients with CM-AVM and lymphatic anomalies. METHODS: Following IRB approval, we identified patients with CM-AVM and lymphatic anomalies seen at the Vascular Anomalies Center at Boston Children's Hospital from 2003 to 2023. We retrospectively reviewed records for clinical, genetic, laboratory, and imaging findings. RESULTS: We found seven patients with CM-AVM and lymphatic abnormalities. Five patients were diagnosed prenatally: four with pleural effusions (including one suspected chylothorax) and one with ascites. Pleural effusions resolved after neonatal drainage in three patients and fetal thoracentesis in the fourth; however, fluid rapidly reaccumulated in this fetus causing hydrops. Ascites resolved after neonatal paracentesis, recurred at 2 months, and spontaneously resolved at 5 years; magnetic resonance lymphangiography for recurrence at age 19 years suggested a central conducting lymphatic anomaly (CCLA), and at age 20 years a right spermatic cord/scrotal lymphatic malformation (LM) was detected. Chylous pericardial effusion presented in a sixth patient at 2 months and disappeared after pericardiocentesis. A seventh patient was diagnosed with a left lower extremity LM at 16 months. Six patients underwent genetic testing, and all had RASA1 mutation. RASA1 variant was novel in three patients (c.1495delinsCTACC, c.434_451delinsA, c.2648del), previously reported in two (c.2603+1G>A, c.475_476del), and unavailable in another. Median follow-up age was 5.8 years (4 months-20 years). CONCLUSION: CM-AVM may be associated with lymphatic anomalies, including pericardial/pleural effusions, ascites, CCLA, and LM.

Clear Cell Squamous Cell Carcinoma of the Maxillary Gingiva Associated with PIK3CA and HRAS Mutations: Report of a Case and Literature Review.

BACKGROUND: Squamous cell carcinoma (SCC) is the most common oral malignancy, and somatic mutations in some driver genes have been implicated in SCC development. Clear cell SCC (CCSCC) is a rare histological variant of SCC, and various clear cell neoplasms must be considered in the differential diagnosis of CCSCC in the oral cavity. Based on a limited number of CCSCC cases reported in the oral cavity, CCSCC is considered an aggressive variant of SCC with a poor prognosis; however, its genetic characteristics remain unknown. METHODS: A maxillary gingival tumor in an 89-year-old female was described and investigated using immunohistochemical staining, special staining, fluorescence in situ hybridization, and next-generation sequencing (NGS) with a custom panel of driver genes, including those associated with SCC and clear cell neoplasm development. RESULTS: Histopathological examination revealed a proliferation of atypical epithelial cells with abundant clear cytoplasm and enlarged and centrally placed round nuclei. The tumor was exophytic with deep, penetrating proliferation. The atypical clear cells were continuous with the conventional SCC cells. Immunohistochemical analysis showed that the clear cells were positive for CK AE1/AE3 and CK5/6 and nuclear-positive for p63. In contrast, the clear cells were negative for αSMA, S100, HMB45, Melan-A, CD10, and p16. p53 immunoreactivity exhibited a wild-type expression pattern. Additionally, the clear cells were positive for periodic acid-Schiff (PAS) and negative for diastase-PAS, mucicarmine, and Alcian blue. Based on these results, the diagnosis of CCSCC was confirmed. Molecular analysis of the clear cells identified PIK3CA p.E542K (c.1624G>A) and HRAS p.G12A (c.35 G>C) somatic mutations classified as oncogenic. No pathogenic variants were identified in TP53, EWSR1, AKT1, PTEN, BRAF, KRAS, NRAS, RASA1, or MAML2. CONCLUSIONS: We report a case of CCSCC of the oral cavity with PIK3CA and HRAS mutations. The identification of PIK3CA and/or HRAS mutations is rare in SCC; however, both mutations are important potential targets for antitumor therapy. A detailed analysis of gene mutations in CCSCC may lead to a better understanding of its biological behavior and an improved prognosis, as well as a differential diagnosis from other clear cell neoplasms.

High-depth next-generation sequencing panel testing in the evaluation of arteriovenous malformations.

Arteriovenous malformations (AVMs) are vascular lesions in which an overgrowth of blood vessels of varying sizes develops with one or more direct connections between the arterial and venous circulation. We performed a retrospective review of a cohort of 54 patients with AVMs referred to our clinical genomic laboratory for high-depth next-generation sequencing (NGS) panel of Disorders of Somatic Mosaicism (DoSM). Thirty-seven of 54 patients were female (68.5%). Among the 54 cases, 37 (68.5%) cases had pathogenic and/or likely pathogenic (P/LP) variants identified, two cases (3.7%) had variants of uncertain clinical significance, and the remaining 15 cases (27.8%) had negative results. MAP2K1 variants were found in 12 cases, followed by eight cases with KRAS variants and seven with TEK variants, and the remainder being identified in several other genes on the panel. Among the 37 positive cases, 32 cases had somatic alterations only; the remaining five cases had at least one germline P/LP variant, including four cases with PTEN and one with RASA1. Of note, two cases had the unexpected co-existence of two P/LP variants. In summary, this study illustrated the molecular diagnostic yield (68.5%) of this cohort of patients with a clinical indication of AVMs by our high-depth DoSM NGS panel.

Prenatal Clinical Findings in RASA1-Related Capillary Malformation-Arteriovenous Malformation Syndrome.

Pathogenic variants in RASA1 are typically associated with a clinical condition called "capillary malformation-arteriovenous malformation" (CM-AVM) syndrome, an autosomal dominant genetic disease characterized by a broad phenotypic variability, even within families. In CM-AVM syndrome, multifocal capillary and arteriovenous malformations are mainly localized in the central nervous system, spine and skin. Although CM-AVM syndrome has been widely described in the literature, only 21 cases with prenatal onset of clinical features have been reported thus far. Here, we report four pediatric cases of molecularly confirmed CM-AVM syndrome which manifested during the prenatal period. Polyhydramnios, non-immune hydrops fetalis and chylothorax are only a few possible aspects of this condition, but a correct interpretation of these prenatal signs is essential due to the possible fatal consequences of unrecognized encephalic and thoracoabdominal deep vascular malformations in newborns and in family members carrying the same RASA1 variant.

Genetic Profile of Arteriovenous Anomalies of the Head and Neck: Implications in Progression and Therapeutic Approaches.

BACKGROUND: Arteriovenous Malformations (AVMs) are complex vascular anomalies that are usually sporadic and can have a variable clinical course. Treatment of AVMs can lead to severe sequeale and require thorough decision-making. There is a lack of standardized treatment protocols showing a growing need for pharmacological targeted therapies, specially in the most severe cases where surgery may not be feasible. Current knowledge in molecular pathways and genetic diagnosis have shed light in the pathophysiology of AVMs, opening possibilities for personalized treatment strategies. METHODS: We performed a retrospective review of patients with head and neck AVMs treated in our department between 2003 and 2021 and performed a complete physical examination and imaging with ultrasound and angio-CT or MRI. Patients underwent genetic testing on AVMs' tissue samples and/or peripheral blood samples. Patients were grouped according to the genetic variant and a correlation between phenotype and genotype was studied. RESULTS: 22 patients with head and neck AVMs were included. We found eight patients with varians in MAP2K1, four patients with pathogenic variants in KRAS, six patients with pathogenic variants in RASA1, one patient with a pathogenic variant in BRAF, one patient with a pathogenic variant in NF1, another patient with a pathogenic variant in CELSR1 and one patient with pathogenic variants in PIK3CA and GNA14. Patients with MAP2K1 variants were the biggest group, with a moderate clinical course. Patients with KRAS mutations showed the most aggressive clinical course and a high rate of recurrence and osteolysis. Patients with RASA1 variants showed a characteristic phenotype with an ipsilateral capillary malformation in the neck. CONCLUSION: We found a correlation between genotype and phenotype in this group of patients. The genetic diagnosis of AVMs is recommended in order to stablish a personalized treatment strategy. Targeted therapies are currently being investigated with promising results and may be recommended in addition to conventional surgical or embolization procedures, specially in the most complex cases. LEVEL OF EVIDENCE: Level IV.

Mutational analysis of microsatellite-stable gastrointestinal cancer with high tumour mutational burden: a retrospective cohort study.

BACKGROUND: Genomic signatures contributing to high tumour mutational burden (TMB-H) independent from mismatch-repair deficiency (dMMR) or microsatellite instability-high (MSI-H) status are not well studied. We aimed to characterise molecular features of microsatellite stable (MSS) TMB-H gastrointestinal tumours. METHODS: Molecular alterations of 48 606 gastrointestinal tumours from Caris Life Sciences (CARIS) identified with next-generation sequencing were compared among MSS-TMB-H, dMMR/MSI-H, and MSS-TMB-low (L) tumours, using χ2 or Fisher's exact tests. Antitumour immune response within the tumour environment was predicted by analysing the infiltration of immune cells and immune signatures using The Cancer Genome Atlas database. The Kaplan-Meier method and the log-rank test were used to evaluate the impact of gene alterations on the efficacy of immune checkpoint inhibitors in MSS gastrointestinal cancers from the CARIS database, a Memorial Sloan Kettering Cancer Center cohort, and a Peking University Cancer Hospital cohort. FINDINGS: MSS-TMB-H was observed in 1600 (3·29%) of 48 606 tumours, dMMR/MSI-H in 2272 (4·67%), and MSS-TMB-L in 44 734 (92·03%). Gene mutations in SMAD2, MTOR, NFE2L2, RB1, KEAP1, TERT, and RASA1 might impair antitumour immune response despite TMB-H, while mutations in 16 other genes (CDC73, CTNNA1, ERBB4, EZH2, JAK2, MAP2K1, MAP2K4, PIK3R1, POLE, PPP2R1A, PPP2R2A, PTPN11, RAF1, RUNX1, STAG2, and XPO1) were related to TMB-H with enhanced antitumour immune response independent of dMMR/MSI-H, constructing a predictive model (modified TMB [mTMB]) for immune checkpoint inhibitor efficacy. Patients with any mutation in the mTMB gene signature, in comparison with patients with mTMB wildtype tumours, showed a superior survival benefit from immune checkpoint inhibitors in MSS gastrointestinal cancers in the CARIS cohort (n=95, median overall survival 18·77 months [95% CI 17·30-20·23] vs 7·03 months [5·73-8·34]; hazard ratio 0·55 [95% CI 0·31-0·99], p=0·044). In addition, copy number amplification in chromosome 11q13 (eg, CCND1, FGF genes) was more prevalent in MSS-TMB-H tumours than in the dMMR/MSI-H or MSS-TMB-L subgroups. INTERPRETATION: Not all mutations related to TMB-H can enhance antitumour immune response. More composite biomarkers should be investigated (eg, mTMB signature) to tailor treatment with immune checkpoint inhibitors. Our data also provide novel insights for the combination of immune checkpoint inhibitors and drugs targeting cyclin D1 or FGFs. FUNDING: US National Cancer Institute, Gloria Borges WunderGlo Foundation, Dhont Family Foundation, Gene Gregg Pancreas Research Fund, San Pedro Peninsula Cancer Guild, Daniel Butler Research Fund, Victoria and Philip Wilson Research Fund, Fong Research Project, Ming Hsieh Research Fund, Shanghai Sailing Program, China National Postdoctoral Program for Innovative Talents, China Postdoctoral Science Foundation, National Natural Science Foundation of China.

Germline genetic mutations in pediatric cerebrovascular anomalies: a multidisciplinary approach to screening, testing, and management.

OBJECTIVE: Genetic alterations are increasingly recognized as etiologic factors linked to the pathogenesis and development of cerebrovascular anomalies. Their identification allows for advanced screening and targeted therapeutic approaches. The authors aimed to describe the role of a collaborative approach to care and genetic testing in pediatric patients with neurovascular anomalies, with the objectives of identifying what genetic testing recommendations were made, the yield of genetic testing, and the implications for familial screening and management at present and in the future. METHODS: The authors performed a descriptive retrospective cohort study examining pediatric patients genetically screened through the Pediatric Neurovascular Program of a single treatment center. Patients 18 years of age and younger with neurovascular anomalies, diagnosed radiographically or histopathologically, were evaluated for germline genetic testing. Patient demographic data and germline genetic testing and recommendation, clinical, treatment, and outcome data were collected and analyzed. RESULTS: Sixty patients were included; 29 (47.5%) were female. The mean age at consultation was 11.0 ± 4.9 years. Diagnoses included cerebral arteriovenous malformations (AVMs) (n = 23), cerebral cavernous malformations (n = 19), non-neurofibromatosis/non-sickle cell moyamoya (n = 8), diffuse cerebral proliferative angiopathy, and megalencephaly-capillary malformation. Of the 56 patients recommended to have genetic testing, 40 completed it. Genetic alterations were found in 13 (23%) patients. Four patients with AVMs had RASA1, GDF2, and ACVRL1 mutations. Four patients with cavernous malformations had Krit1 mutations. One with moyamoya disease had an RNF213 mutation. Three patients with megalencephaly-capillary malformation had PIK3CA mutations, and 1 patient with a cavernous sinus lesion had an MED12 mutation. The majority of AVM patients were treated surgically. Patients with diffuse cerebral proliferative angiopathy were treated medically with sirolimus. At-risk relatives of 3 patients positive for genetic anomalies had also been tested. CONCLUSIONS: This study demonstrates a role for exploring genetic alterations in the identification and treatment of pediatric neurovascular disease pathogenesis. Germline genetic mutations were found in almost one-quarter of the patients screened in this study, results that helped to identify medically targeted treatment modalities for some pediatric neurovascular patients. Insight into the genetic etiology of vascular anomalies may provide broader clinical implications for risk assessment, family screening, follow-up surveillance, and medical management.

Genetic testing in the evaluation of individuals with clinical diagnosis of atypical Sturge-Weber syndrome.

Sturge-Weber Syndrome (SWS) is a rare vascular malformation disorder characterized by abnormal blood vessels in the brain, skin, and eye. SWS is most commonly caused by a somatic mosaic GNAQ-p.Arg183Gln variant. In this series, 12 patients presented for clinical evaluation of SWS but were noted to have atypical features, and therefore germline and/or somatic genetic testing was performed. Atypical features included extensive capillary malformation on the body as well as the face, frontal bossing, macrocephaly, telangiectasia, overgrowth of extremities, absence of neurologic signs and symptoms, and family history of vascular malformations. Five patients had a somatic GNAQ or GNA11 pathogenic variant, one patient had a somatic mosaic likely-pathogenic variant in PIK3CA, and another one had a somatic mosaic deletion that disrupted PTPRD. The other five patients had germline variants in RASA1, EPHB4, or KIT. Our findings suggest that patients presenting for SWS evaluation who have atypical clinical characteristics may have pathogenic germline or somatic variants in genes other than GNAQ or GNA11. Broad germline and somatic genetic testing in these patients with atypical findings may have implications for medical care, prognosis, and trial eligibility.

Genotypes and phenotypes heterogeneity in PIK3CA-related overgrowth spectrum and overlapping conditions: 150 novel patients and systematic review of 1007 patients with PIK3CA pathogenetic variants.

BACKGROUND: Postzygotic activating PIK3CA variants cause several phenotypes within the PIK3CA-related overgrowth spectrum (PROS). Variant strength, mosaicism level, specific tissue involvement and overlapping disorders are responsible for disease heterogeneity. We explored these factors in 150 novel patients and in an expanded cohort of 1007 PIK3CA-mutated patients, analysing our new data with previous literature to give a comprehensive picture. METHODS: We performed ultradeep targeted next-generation sequencing (NGS) on DNA from skin biopsy, buccal swab or blood using a panel including phosphatidylinositol 3-kinase/AKT/mammalian target of rapamycin pathway genes and GNAQ, GNA11, RASA1 and TEK. Additionally, 914 patients previously reported were systematically reviewed. RESULTS: 93 of our 150 patients had PIK3CA pathogenetic variants. The merged PROS cohort showed that PIK3CA variants span thorough all gene domains, some were exclusively associated with specific PROS phenotypes: weakly activating variants were associated with central nervous system (CNS) involvement, and strongly activating variants with extra-CNS phenotypes. Among the 57 with a wild-type PIK3CA allele, 11 patients with overgrowth and vascular malformations overlapping PROS had variants in GNAQ, GNA11, RASA1 or TEK. CONCLUSION: We confirm that (1) molecular diagnostic yield increases when multiple tissues are tested and by enriching NGS panels with genes of overlapping 'vascular' phenotypes; (2) strongly activating PIK3CA variants are found in affected tissue, rarely in blood: conversely, weakly activating mutations more common in blood; (3) weakly activating variants correlate with CNS involvement, strong variants are more common in cases without; (4) patients with vascular malformations overlapping those of PROS can harbour variants in genes other than PIK3CA.

Prognostic features and comprehensive genomic analysis of NF1 mutations in EGFR mutant lung cancer patients.

OBJECTIVE: NF1 is a tumor suppressor gene that encodes the neurofibromin protein and negatively regulates Ras signaling. This study was aimed to investigate the molecular, clinical characteristics, and prognostic features of NF1 gene in EGFR mutant lung cancer patients. METHOD: The next-generation sequencing (NGS) was used to analyze the data from lung cancer patients in the Guangdong Lung Cancer Institute (GLCI) from June 2016 to December 2020. RESULTS: Somatic NF1 mutations were present in 4.2% (135/3220) of Chinese lung cancer patients. NF1 mutations where clearly enriched in older (p < 0.001), male (p < 0.001), and smoking (p < 0.001) patients. Patients with NF1 mutations were more likely to have TP53 (p = 0.003), BRAF (p = 0.001) and RASA1 (p = 0.026) mutations and mutually exclusive with EGFR mutations (p = 0.006). TP53 mutation had worsen prognosis in cases of NF1 mutant (p = 0.026) or EGFR/NF1 co-mutant (p = 0.031) lung adenocarcinomas (LUAD) patients. There was no effect on overall survival (OS) in LUAD patients with and without NF1 mutations, even in LUAD driver-gene negative patients. NF1/EGFR co-mutation patients had a longer OS than a single mutation of either the EGFR gene (median OS: 47.7 m vs. 30.2 m, hazard ratio [95% CI], 0.47 [0.30-0.74], p = 0.004) or NF1 gene (47.7 m vs. 19.0 m, 0.44 [0.27-0.73], p = 0.003). Furthermore, NF1 mutations significantly prolonged OS in EGFR mutant/TP53 wild-type LUAD patients (106.5 m vs. 25.5 m, 0.28 [0.13-0.59], p = 0.003) but not in patients with EGFR/TP53 co-mutations (36.8 m vs. 30.2 m, 0.70 [0.39-1.26], p = 0.280). CONCLUSION: Our results indicated NF1 mutations served as a good prognostic factor in EGFR mutant/TP53 wild-type lung cancer patients in this single-center study. TP53 mutation was obviously enriched in NF1 mutant patients and had shorter OS.

Three Cases of Familial Moyamoya Disease with RASA1 Mutations-A Case Report.

OBJECTIVE: The prevalence of moyamoya disease (MMD) is relatively high in East Asia, whereas the susceptibility genes of MMD have not been identified. METHODS: Here, we reported 3 patients diagnosed with MMD from 1 single family, including a 53-year-old mother (case 1) and her 32-year-old and 29-year-old daughters (cases 2 and 3). The younger daughter was diagnosed with cerebral hemorrhage. Computed tomographic angiogram showed the typical signs of MMD in 3 patients. RESULTS: Clinical whole-exome sequencing was performed in 3 daughters of case 1, and RASA1 mutations in chr5: 87,376,389 and NM_002890.2: c.2012-4C > T were determined to have the strongest correlation with MMD. RASA1 mutations were verified in case 1, husband of case 1 and the descendant of case 3 by using Sanger sequencing. CONCLUSIONS: According to the findings of literature review, this is the first study indicating the association between RASA1 mutations and MMD.

Regulation of IncRNA ZNF667-AS1 in Proliferation and Invasion of Esophageal Squamous Cell Carcinoma Cells via Mediating ceRNA Network.

Esophageal squamous cell carcinoma (ESCC), classified as a primary histological subtype of esophageal cancer (EC), dominates approximately 90% of the newly diagnosed EC. Long non-coding RNAs (lncRNAs) are frequently related to the course of ESCC. The current study aimed to investigate whether lncRNA zinc finger protein 667-antisense RNA 1 (ZNF667-AS1) modulates the proliferation and invasion of ESCC cells. ESCC tissues and cell lines, para-carcinoma tissues, and human esophageal epithelial cells (HEEpiCs) were collected. lncRNA ZNF667-AS1 expression in the above tissues and cells was detected. The effect of lncRNA ZNF667-AS1 on proliferation and invasion of Eca109 cells was detected using cell counting kit-8, colony formation, and Transwell assays. lncRNA ZNF667-AS1 subcellular localization was determined via the nuclear/cytosol fractionation assay. The binding relationships between miR-18b-5p and lncRNA ZNF667-AS1 and RAS p21 protein activator 1 (RASA1) were verified using dual-luciferase reporter gene experiment and RNA immunoprecipitation experiment. The expressions of miR-18b-5p and RASA1 in the tissues and cells were identified. The roles of miR-18b-5p overexpression or silencing RASA1 in proliferation and invasion of ESCC cells were examined through rescue experiments. lncRNA ZNF667-AS1 was underexpressed in ESCC tissues and cells, and lncRNA ZNF667-AS1 overexpression hampered ESCC cell proliferation and invasiveness. miR-18b-5p targeted RASA1 while lncRNA ZNF667-AS1 promoted RASA1 transcription via binding to miR-18b-5p. Over-expression miR-18b-5p or silencing RASA1 reversed the inhibitory effects of lncRNA ZNF667-AS1 overexpression on ESCC cell proliferation and invasion. lncRNA ZNF667-AS1 overexpression accelerated RASA1 transcription by competitively binding to miR-18b-5p, thus suppressing ESCC cell proliferation and invasion.

SH3 domain regulation of RhoGAP activity: Crosstalk between p120RasGAP and DLC1 RhoGAP.

RhoGAP proteins are key regulators of Rho family GTPases and influence a variety of cellular processes, including cell migration, adhesion, and cytokinesis. These GTPase activating proteins (GAPs) downregulate Rho signaling by binding and enhancing the intrinsic GTPase activity of Rho proteins. Deleted in liver cancer 1 (DLC1) is a tumor suppressor and ubiquitously expressed RhoGAP protein; its activity is regulated in part by binding p120RasGAP, a GAP protein for the Ras GTPases. In this study, we report the co-crystal structure of the p120RasGAP SH3 domain bound directly to DLC1 RhoGAP, at a site partially overlapping the RhoA binding site and impinging on the catalytic arginine finger. We demonstrate biochemically that mutation of this interface relieves inhibition of RhoGAP activity by the SH3 domain. These results reveal the mechanism for inhibition of DLC1 RhoGAP activity by p120RasGAP and demonstrate the molecular basis for direct SH3 domain modulation of GAP activity.

RASopathies due to de novo pathogenic variants: clinical features, genetic findings and outcomes in nine neonates born with congenital heart defects.

BACKGROUND: There are limited information available related to neonatal characteristics of RASopathies, a group of autosomal dominant syndromes with considerable phenotypic overlap. METHODS: The retrospective review revealed 9 neonates born with congenital heart defects (CHDs) and diagnosed as RASopathies due to de novo mutations (DNMs) by trio-based exome sequencing (ES) between January 2017 and December 2020. We report in details of the neonatal course, molecular analysis and 180-days of age follow-up in affected individuals. RESULTS: The early clinical spectrum included various types of CHDs, less noticeable multiple extracardiac anomalies and unspecific symptoms like poor feeding. Of the 8 variants identified from 6 genes, 2 in RASA1 were novel: (NM_002890.2: c.2828 T > C (p.Leu943Pro)) and (NM_002890.2: c.2001del (p.Pro668Leufs*10)), which functionally impaired the protein structure. There was a relatively high mortality rate of 33.33% (3/9) for all the defects combined. A RAF1-deficient male and a RASA1-deficient male survived from severe heart failure by surgical interventions in early life. CONCLUSIONS: Our results revealed that family-based ES was useful in identifying DNMs and causal genes for sporadic diseases and screening Rasopathies shortly after birth. We recommended a family-based ES and a full phenotypic evaluation including echocardiogram, magnetic resonance imaging, ultrasonography and coagulation screening in neonates with CHDs and a suspected genetic etiology.

Capillary malformation-arteriovenous malformation syndrome associated with basilar artery aneurysm.

A 23-day-old boy with prenatal diagnosis of basilar artery aneurysm presented with multiple congenital red patches consistent with capillary malformations. Genetic testing confirmed the presence of a heterozygous pathogenic variant of the RASA1 gene, confirming the diagnosis of capillary malformation-arteriovenous malformation (CM-AVM) syndrome. This case illustrates an atypical presentation of the RASA1 associated CM-AVM syndrome, with the intracranial vascular malformation diagnosis preceding the identification of the skin lesions. Arterial aneurysms have been associated with CM-AVM syndrome in rare instances but to our knowledge this is the first reported case of an aneurysm of the basilar artery.

MicroRNA-4487 regulates vascular smooth muscle cell proliferation, migration and apoptosis by targeting RAS p21 protein activator 1.

BACKGROUND: Dysregulation of microRNA (miRNA) is involved in the pathogenesis of a variety of diseases, including atherosclerosis (AS). However, the role of miRNA-4487 (miR-4487) in the development of AS is not fully clarified. This study is intended to investigate the regulatory effects of miR-4487 on the proliferation, migration and apoptosis of vascular smooth muscle cells (VSMCs) and the related mechanisms. METHODS: Oxidized low-density lipoprotein (ox-LDL) was employed to induce the dysfunction of VSMCs. Subsequently, miR-4487 expression was detected by quantitative real-time PCR. Afterward, the expression levels of RAS p21 protein activator 1 (RASA1) and apoptosis-related proteins (Bcl-2, Bax, Cleaved caspase 3, Cleaved caspase 9) were detected by Western blotting. The proliferation, migration and apoptosis of VSMCs were then detected by CCK-8, BrdU, Transwell and flow cytometry assays, respectively. Moreover, a dual-luciferase reporter gene assay was executed to verify the targeting between miR-4487 to the RASA1 3'-untranslated region (3'-UTR). RESULTS: ox-LDL treatment increased miR-4487 expression and decreased RASA1 expression in VSMCs. Additionally, ox-LDL treatment promoted the proliferation and migration of VSMCs, but inhibited apoptosis. Besides, the effects of ox-LDL treatment on the proliferation, migration and apoptosis of VSMCs were attenuated by the transfection of miR-4487 inhibitors. Furthermore, miR-4487 directly targeted the 3'-UTR of RASA1 mRNA and repressed the expression level of RASA1. Also, RASA1 knockdown reversed the effects of miR-4487 inhibition on VSMCs. CONCLUSION: MiR-4487 promotes VSMCs viability and migration and inhibits apoptosis by targeting RASA1 in VSMCs, by which it promotes the pathogenesis of AS.