DLG2 intragenic exonic deletions reinforce the link to neurodevelopmental disorders and suggest a potential association with congenital anomalies and dysmorphism.

PURPOSE: Multiple studies suggest an association between DLG2 and neurodevelopmental disorders and indicate the haploinsufficiency of this gene; however, few cases have been thoroughly described. We performed additional studies to confirm this clinical association and DLG2 haploinsufficiency. METHODS: Chromosomal microarray analysis was performed on 11,107 patients at the Cytogenetics Laboratory at the University of Alabama at Birmingham. The Database of Genomic Variants-Gold Standard Variants and the Genome Aggregation Database were selected for the association analysis. Fifty-nine patients from the literature and DECIPHER, all having DLG2 intragenic deletions, were included for comprehensive analysis of the distribution of these deletions. RESULTS: A total of 13 patients with DLG2 intragenic deletions, from 10 families in our cohort, were identified. Nine of 10 probands presented with clinical features of neurodevelopmental disorders. Congenital anomalies and dysmorphism were common in our cohort of patients. Association analysis showed that the frequency of DLG2 deletions in our cohort is significantly higher than those in the Database of Genomic Variants-Gold Standard Variants and the Genome Aggregation Database. Most of DLG2 intragenic deletions identified in 69 unrelated patients from our cohort, the literature, and DECIPHER map to the 5' region of the gene, with a hotspot centered around HPin7, exon 8, and HPin8. CONCLUSION: Our findings reinforce the link between DLG2 intragenic deletions and neurodevelopmental disorders, strongly support the haploinsufficiency of this gene, and indicate that these deletions might also have an association with congenital anomalies and dysmorphism.

Epigenetic switch reshapes epithelial progenitor cell signatures and drives inflammatory pathogenesis in hidradenitis suppurativa.

Hidradenitis suppurativa (HS) is a complex inflammatory skin disease with undefined mechanistic underpinnings. Here, we investigated HS epithelial cells and demonstrated that HS basal progenitors modulate their lineage restriction and give rise to pathogenic keratinocyte clones, resulting in epidermal hyperproliferation and dysregulated inflammation in HS. When comparing to healthy epithelial stem/progenitor cells, in HS, we identified changes in gene signatures that revolve around the mitotic cell cycle, DNA damage response and repair, as well as cell-cell adhesion and chromatin remodeling. By reconstructing cell differentiation trajectory and CellChat modeling, we identified a keratinocyte population specific to HS. This population is marked by S100A7/8/9 and KRT6 family members, triggering IL1, IL10, and complement inflammatory cascades. These signals, along with HS-specific proinflammatory cytokines and chemokines, contribute to the recruitment of certain immune cells during the disease progression. Furthermore, we revealed a previously uncharacterized role of S100A8 in regulating the local chromatin environment of target loci in HS keratinocytes. Through the integration of genomic and epigenomic datasets, we identified genome-wide chromatin rewiring alongside the switch of transcription factors (TFs), which mediated HS transcriptional profiles. Importantly, we identified numerous clinically relevant inflammatory enhancers and their coordinated TFs in HS basal CD49fhigh cells. The disruption of the S100A enhancer using the CRISPR/Cas9-mediated approach or the pharmacological inhibition of the interferon regulatory transcription factor 3 (IRF3) efficiently reduced the production of HS-associated inflammatory regulators. Our study not only uncovers the plasticity of epidermal progenitor cells in HS but also elucidates the epigenetic mechanisms underlying HS pathogenesis.

Mechanism underlying follicular hyperproliferation and oncogenesis in hidradenitis suppurativa.

Hidradenitis suppurativa (HS) is a skin disorder that causes chronic painful inflammation and hyperproliferation, often with the comorbidity of invasive keratoacanthoma (KA). Our research, employing high-resolution immunofluorescence and data science approaches together with confirmatory molecular analysis, has identified that the 5'-cap-dependent protein translation regulatory complex eIF4F is a key factor in the development of HS and is responsible for regulating follicular hyperproliferation. Specifically, eIF4F translational targets, Cyclin D1 and c-MYC, orchestrate the development of HS-associated KA. Although eIF4F and p-eIF4E are contiguous throughout HS lesions, Cyclin D1 and c-MYC have unique spatial localization and functions. The keratin-filled crater of KA is formed by nuclear c-MYC-induced differentiation of epithelial cells, whereas the co-localization of c-MYC and Cyclin D1 provides oncogenic transformation by activating RAS, PI3K, and ERK pathways. In sum, we have revealed a novel mechanism underlying HS pathogenesis of follicular hyperproliferation and the development of HS-associated invasive KA.

Analysis of 200 unrelated individuals with a constitutional NF1 deep intronic pathogenic variant reveals that variants flanking the alternatively spliced NF1 exon 31 [23a] cause a classical neurofibromatosis type 1 phenotype while altering predominantly NF1 isoform type II.

Neurofibromatosis type 1 results from loss-of-function NF1 pathogenic variants (PVs). Up to 30% of all NF1 PVs disrupt mRNA splicing, including deep intronic variants. Here, we retrospectively investigated the spectrum of NF1 deep intronic PVs in a cohort of 8,090 unrelated individuals from the University of Alabama at Birmingham (UAB) dataset with a molecularly confirmed neurofibromatosis type 1. All variants were identified through their effect on the NF1 transcript, followed by variant characterization at the DNA-level. A total of 68 distinct variants, which were ≥ 20 nucleotides away from the closest exon-intron junction, were identified in 2.5% unrelated individuals with NF1 (200/8,090). Nine different pathogenic splice variants, identified in 20 probands, led to exonization of different parts of intron 30 [23.2] or 31 [23a]. The two major NF1 transcript isoforms, distinguished by the absence (type I) or presence (type II) of the alternatively spliced cassette exon 31 [23a], are equally expressed in blood in control individuals without NF1 or NF1-affected individuals carrying their PV not in the introns flanking exon 31 [23a]. By fragment and cloning analysis we demonstrated that the exonization of intron 31 [23a] sequences due to deep intronic PV predominantly affects the NF1 isoform II. Seven additional (likely) pathogenic NF1 deep intronic variants not observed in the UAB dataset were found by classification of 36 variants identified by a literature search. Hence, the unique list of these 75 deep intronic (likely) PVs should be included in any comprehensive NF1 testing strategy.

Three-dimensional analysis of the relationship between mandibular retromolar space and positional traits of third molars in non-hyperdivergent adults.

BACKGROUND: The anatomical position of the mandibular third molars (M3s) is located in the distal-most portions of the molar area. In some previous literature, researchers evaluated the relationship between retromolar space (RS) and different classifications of M3 in three­dimensional (3D) cone-beam computed tomography (CBCT). METHODS: Two hundred six M3s from 103 patients were included. M3s were grouped according to four classification criteria: PG-A/B/C, PG-I/II/III, mesiodistal angle and buccolingual angle. 3D hard tissue models were reconstructed by CBCT digital imaging. RS was measured respectively by utilizing the fitting WALA ridge plane (WP) which was fitted by the least square method and the occlusal plane (OP) as reference planes. SPSS (version 26) was used to analyze the data. RESULTS: In all criteria evaluated, RS decreased steadily from the crown to the root (P < 0.05), the minimum was at the root tip. From PG-A classification, PG-B classification to PG-C classification and from PG-I classification, PG-II classification to PG-III classification, RS both appeared a diminishing tendency (P < 0.05). As the degree of mesial tilt decreased, RS appeared an increasing trend (P < 0.05). RS in classification criteria of buccolingual angle had no statistical difference (P > 0.05). CONCLUSIONS: RS was associated with positional classifications of the M3. In the clinic, RS can be evaluated by watching the Pell&Gregory classification and mesial angle of M3.

Activation of a novel α2AAR-spinophilin-cofilin axis determines the effect of α2 adrenergic drugs on fear memory reconsolidation.

Posttraumatic stress disorder (PTSD) after the pandemic has emerged as a major neuropsychiatric component of post-acute COVID-19 syndrome, yet the current pharmacotherapy for PTSD is limited. The use of adrenergic drugs to treat PTSD has been suggested; however, it is hindered by conflicting clinical results and a lack of mechanistic understanding of drug actions. Our studies, using both genetically modified mice and human induced pluripotent stem cell-derived neurons, reveal a novel α2A adrenergic receptor (α2AAR)-spinophilin-cofilin axis in the hippocampus that is critical for regulation of contextual fear memory reconsolidation. In addition, we have found that two α2 ligands, clonidine and guanfacine, exhibit differential abilities in activating this signaling axis to disrupt fear memory reconsolidation. Stimulation of α2AAR with clonidine, but not guanfacine, promotes the interaction of the actin binding protein cofilin with the receptor and with the dendritic spine scaffolding protein spinophilin to induce cofilin activation at the synapse. Spinophilin-dependent regulation of cofilin is required for clonidine-induced disruption of contextual fear memory reconsolidation. Our results inform the interpretation of differential clinical observations of these two drugs on PTSD and suggest that clonidine could provide immediate treatment for PTSD symptoms related to the current pandemic. Furthermore, our study indicates that modulation of dendritic spine morphology may represent an effective strategy for the development of new pharmacotherapies for PTSD.

A peptide blocking the ADORA1-neurabin interaction is anticonvulsant and inhibits epilepsy in an Alzheimer's model.

Epileptic seizures are common sequelae of stroke, acute brain injury, and chronic neurodegenerative diseases, including Alzheimer's disease (AD), and cannot be effectively controlled in approximately 40% of patients, necessitating the development of novel therapeutic agents. Activation of the A1 receptor (A1R) by endogenous adenosine is an intrinsic mechanism to self-terminate seizures and protect neurons from excitotoxicity. However, targeting A1R for neurological disorders has been hindered by side effects associated with its broad expression outside the nervous system. Here we aim to target the neural-specific A1R/neurabin/regulator of G protein signaling 4 (A1R/neurabin/RGS4) complex that dictates A1R signaling strength and response outcome in the brain. We developed a peptide that blocks the A1R-neurabin interaction to enhance A1R activity. Intracerebroventricular or i.n. administration of this peptide shows marked protection against kainate-induced seizures and neuronal death. Furthermore, in an AD mouse model with spontaneous seizures, nasal delivery of this blocking peptide reduces epileptic spike frequency. Significantly, the anticonvulsant and neuroprotective effects of this peptide are achieved through enhanced A1R function in response to endogenous adenosine in the brain, thus, avoiding side effects associated with A1R activation in peripheral tissues and organs. Our study informs potentially new anti-seizure therapy applicable to epilepsy and other neurological illness with comorbid seizures.

Targeted massively parallel sequencing of candidate regions on chromosome 22q predisposing to multiple schwannomas: An analysis of 51 individuals in a single-center experience.

Constitutional LZTR1 or SMARCB1 pathogenic variants (PVs) have been found in ∼86% of familial and ∼40% of sporadic schwannomatosis cases. Hence, we performed massively parallel sequencing of the entire LZTR1, SMARCB1, and NF2 genomic loci in 35 individuals with schwannomas negative for constitutional first-hit PVs in the LZTR1/SMARCB1/NF2 coding sequences; however, with 22q deletion and/or a different NF2 PV in each tumor, including six cases with only one tumor available. Furthermore, we verified whether any other LZTR1/SMARCB1/NF2 (likely) PVs could be found in 16 cases carrying a SMARCB1 constitutional variant in the 3'-untranslated region (3'-UTR) c.*17C>T, c.*70C>T, or c.*82C>T. As no additional variants were found, functional studies were performed to clarify the effect of these 3'-UTR variants on the transcript. The 3'-UTR variants c.*17C>T and c.*82C>T showed pathogenicity by negatively affecting the SMARCB1 transcript level. Two novel deep intronic SMARCB1 variants, c.500+883T>G and c.500+887G>A, resulting in out-of-frame missplicing of intron 4, were identified in two unrelated individuals. Further resequencing of the entire repeat-masked genomics sequences of chromosome 22q in individuals negative for PVs in the SMARCB1/LZTR1/NF2 coding- and noncoding regions revealed five potential schwannomatosis-predisposing candidate genes, that is, MYO18B, NEFH, SGSM1, SGSM3, and SBF1, pending further verification.

STRAP regulates alternative splicing fidelity during lineage commitment of mouse embryonic stem cells.

Alternative splicing (AS) is involved in cell fate decisions and embryonic development. However, regulation of these processes is poorly understood. Here, we have identified the serine threonine kinase receptor-associated protein (STRAP) as a putative spliceosome-associated factor. Upon Strap deletion, there are numerous AS events observed in mouse embryoid bodies (EBs) undergoing a neuroectoderm-like state. Global mapping of STRAP-RNA binding in mouse embryos by enhanced-CLIP sequencing (eCLIP-seq) reveals that STRAP preferably targets transcripts for nervous system development and regulates AS through preferred binding positions, as demonstrated for two neuronal-specific genes, Nnat and Mark3. We have found that STRAP involves in the assembly of 17S U2 snRNP proteins. Moreover, in Xenopus, loss of Strap leads to impeded lineage differentiation in embryos, delayed neural tube closure, and altered exon skipping. Collectively, our findings reveal a previously unknown function of STRAP in mediating the splicing networks of lineage commitment, alteration of which may be involved in early embryonic lethality in mice.

Constitutional mismatch repair deficiency is the diagnosis in 0.41% of pathogenic NF1/SPRED1 variant negative children suspected of sporadic neurofibromatosis type 1.

PURPOSE: Biallelic germline mismatch repair (MMR) gene pathogenic variants (PVs) cause constitutional MMR deficiency (CMMRD), a highly penetrant childhood cancer syndrome phenotypically overlapping with neurofibromatosis type 1 (NF1). CMMRD testing in suspected NF1 children without NF1/SPRED1 PVs enables inclusion of CMMRD positives into monitoring programs prior to tumor onset. However, testing is associated with potential harms and the prevalence of CMMRD among these children is unknown. METHODS: Using a simple and scalable microsatellite instability (MSI) assay of non-neoplastic leukocyte DNA to detect CMMRD, we retrospectively screened >700 children suspected of sporadic NF1 but lacking NF1/SPRED1 PVs. RESULTS: For three of seven MSI-positive patients germline MMR gene PVs confirmed the diagnosis of CMMRD. Founder variants NM_000535.5(PMS2):c.736_741delinsTGTGTGTGAAG, prevalent in Europe and North America, and NM_000179.2(MSH6):c.10C>G, affecting 1:400 French Canadians, represented two of five PVs. The prevalence of CMMRD was 3/735 (0.41%, 95% confidence interval [CI]: 0.08-1.19%). CONCLUSION: Our empirical data provide reliable numbers for genetic counseling and confirm previous prevalence estimations, on which Care for CMMRD consortium guidelines are based. These advocate CMMRD testing of preselected patients rather than offering reflex testing to all suspected sporadic NF1 children lacking NF1/SPRED1 PVs. The possibility of founder effects should be considered alongside these testing guidelines.

AG-exclusion zone revisited: Lessons to learn from 91 intronic NF1 3' splice site mutations outside the canonical AG-dinucleotides.

Uncovering frequent motives of action by which variants impair 3' splice site (3'ss) recognition and selection is essential to improve our understanding of this complex process. Through several mini-gene experiments, we demonstrate that the pyrimidine (Y) to purine (R) transversion NM_000267.3(NF1):c.1722-11T>G, although expected to weaken the polypyrimidine tract, causes exon skipping primarily by introducing a novel AG in the AG-exclusion zone (AGEZ) between the authentic 3'ss AG and the branch point. Evaluation of 90 additional noncanonical intronic NF1 3'ss mutations confirmed that 63% of all mutations and 89% (49/55) of the single-nucleotide variants upstream of positions -3 interrupt the AGEZ. Of these AGEZ-interrupting mutations, 24/49 lead to exon skipping suggesting that absence of AG in this region is necessary for accurate 3'ss selection already in the initial steps of splicing. The analysis of 91 noncanonical NF1 3'ss mutations also shows that 90% either introduce a novel AG in the AGEZ, cause a Y>R transversion at position -3 or remove ≥2 Ys in the AGEZ. We confirm in a validation cohort that these three motives distinguish spliceogenic from splice-neutral variants with 85% accuracy and, therefore, are generally applicable to select among variants of unknown significance those likely to affect splicing.

Author Correction: The in vivo specificity of synaptic Gß and Gγ subunits to the α2a adrenergic receptor at CNS synapses.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

ß-amyloid redirects norepinephrine signaling to activate the pathogenic GSK3ß/tau cascade.

The brain noradrenergic system is critical for normal cognition and is affected at early stages in Alzheimer's disease (AD). Here, we reveal a previously unappreciated direct role of norepinephrine signaling in connecting ß-amyloid (Aß) and tau, two key pathological components of AD pathogenesis. Our results show that Aß oligomers bind to an allosteric site on α2A adrenergic receptor (α2AAR) to redirect norepinephrine-elicited signaling to glycogen synthase kinase 3ß (GSK3ß) activation and tau hyperphosphorylation. This norepinephrine-dependent mechanism sensitizes pathological GSK3ß/tau activation in response to nanomolar accumulations of extracellular Aß, which is 50- to 100-fold lower than the amount required to activate GSK3ß by Aß alone. The significance of our findings is supported by in vivo evidence in two mouse models, human tissue sample analysis, and longitudinal clinical data. Our study provides translational insights into mechanisms underlying Aß proteotoxicity, which might have strong implications for the interpretation of Aß clearance trial results and future drug design and for understanding the selective vulnerability of noradrenergic neurons in AD.

Clinical spectrum of individuals with pathogenic NF1 missense variants affecting p.Met1149, p.Arg1276, and p.Lys1423: genotype-phenotype study in neurofibromatosis type 1.

We report 281 individuals carrying a pathogenic recurrent NF1 missense variant at p.Met1149, p.Arg1276, or p.Lys1423, representing three nontruncating NF1 hotspots in the University of Alabama at Birmingham (UAB) cohort, together identified in 1.8% of unrelated NF1 individuals. About 25% (95% confidence interval: 20.5-31.2%) of individuals heterozygous for a pathogenic NF1 p.Met1149, p.Arg1276, or p.Lys1423 missense variant had a Noonan-like phenotype, which is significantly more compared with the "classic" NF1-affected cohorts (all p < .0001). Furthermore, p.Arg1276 and p.Lys1423 pathogenic missense variants were associated with a high prevalence of cardiovascular abnormalities, including pulmonic stenosis (all p < .0001), while p.Arg1276 variants had a high prevalence of symptomatic spinal neurofibromas (p < .0001) compared with "classic" NF1-affected cohorts. However, p.Met1149-positive individuals had a mild phenotype, characterized mainly by pigmentary manifestations without externally visible plexiform neurofibromas, symptomatic spinal neurofibromas or symptomatic optic pathway gliomas. As up to 0.4% of unrelated individuals in the UAB cohort carries a p.Met1149 missense variant, this finding will contribute to more accurate stratification of a significant number of NF1 individuals. Although clinically relevant genotype-phenotype correlations are rare in NF1, each affecting only a small percentage of individuals, together they impact counseling and management of a significant number of the NF1 population.

The in vivo specificity of synaptic Gß and Gγ subunits to the α2a adrenergic receptor at CNS synapses.

G proteins are major transducers of signals from G-protein coupled receptors (GPCRs). They are made up of α, ß, and γ subunits, with 16 Gα, 5 Gß and 12 Gγ subunits. Though much is known about the specificity of Gα subunits, the specificity of Gßγs activated by a given GPCR and that activate each effector in vivo is not known. Here, we examined the in vivo Gßγ specificity of presynaptic α2a-adrenergic receptors (α2aARs) in both adrenergic (auto-α2aARs) and non-adrenergic neurons (hetero-α2aARs) for the first time. With a quantitative MRM proteomic analysis of neuronal Gß and Gγ subunits, and co-immunoprecipitation of tagged α2aARs from mouse models including transgenic FLAG-α2aARs and knock-in HA-α2aARs, we investigated the in vivo specificity of Gß and Gγ subunits to auto-α2aARs and hetero-α2aARs activated with epinephrine to understand the role of Gßγ specificity in diverse physiological functions such as anesthetic sparing, and working memory enhancement. We detected Gß2, Gγ2, Gγ3, and Gγ4 with activated auto α2aARs, whereas we found Gß4 and Gγ12 preferentially interacted with activated hetero-α2aARs. Further understanding of in vivo Gßγ specificity to various GPCRs offers new insights into the multiplicity of genes for Gß and Gγ, and the mechanisms underlying GPCR signaling through Gßγ subunits.

Correction: Expanding the clinical phenotype of individuals with a 3-bp in-frame deletion of the NF1 gene (c.2970_2972del): an update of genotype-phenotype correlation.

A correction has been published to this Article. The PDF and HTML have been updated accordingly.

Expanding the clinical phenotype of individuals with a 3-bp in-frame deletion of the NF1 gene (c.2970_2972del): an update of genotype-phenotype correlation.

PURPOSE: Neurofibromatosis type 1 (NF1) is characterized by a highly variable clinical presentation, but almost all NF1-affected adults present with cutaneous and/or subcutaneous neurofibromas. Exceptions are individuals heterozygous for the NF1 in-frame deletion, c.2970_2972del (p.Met992del), associated with a mild phenotype without any externally visible tumors. METHODS: A total of 135 individuals from 103 unrelated families, all carrying the constitutional NF1 p.Met992del pathogenic variant and clinically assessed using the same standardized phenotypic checklist form, were included in this study. RESULTS: None of the individuals had externally visible plexiform or histopathologically confirmed cutaneous or subcutaneous neurofibromas. We did not identify any complications, such as symptomatic optic pathway gliomas (OPGs) or symptomatic spinal neurofibromas; however, 4.8% of individuals had nonoptic brain tumors, mostly low-grade and asymptomatic, and 38.8% had cognitive impairment/learning disabilities. In an individual with the NF1 constitutional c.2970_2972del and three astrocytomas, we provided proof that all were NF1-associated tumors given loss of heterozygosity at three intragenic NF1 microsatellite markers and c.2970_2972del. CONCLUSION: We demonstrate that individuals with the NF1 p.Met992del pathogenic variant have a mild NF1 phenotype lacking clinically suspected plexiform, cutaneous, or subcutaneous neurofibromas. However, learning difficulties are clearly part of the phenotypic presentation in these individuals and will require specialized care.

Comparative evaluation of the vertical fracture resistance of endodontically treated roots filled with Gutta-percha and Resilon: a meta-analysis of in vitro studies.

BACKGROUND: Teeth treated endodontically are more susceptible to vertical root fracture (VRF). Some studies have suggested that obturating the root canals with Gutta-percha or Resilon can reinforce endodontically treated teeth, but a few others have presented conflicting results. These inconsistent results cannot guide clinicians in determining clinical approaches. The objective of this meta-analysis is to evaluate and compare the vertical fracture resistance of endodontically treated root canals obturated with Gutta-percha/AH plus and the Resilon system. METHODS: Comprehensive literature searches were performed in the PubMed, Cochrane Library, ScienceDirect, Web of Science and Embase databases. The titles and abstracts of all of the retrieved articles were independently assessed by two authors according to predefined selection criteria. Data in the included articles were independently extracted. Statistical analyses were conducted using Review Manager 5.3 and Stata 12.0 software. The pooled standardized mean differences (SMDs) with 95% confidence intervals (CIs) were calculated for the outcome indicators. The level of statistical significance was set at p < 0.05. The Cochran Q test (I2 test) was used to test for heterogeneity among studies. RESULTS: Fourteen randomized controlled in vitro trials were included in the meta-analysis. The results demonstrated that the vertical root fracture resistance of unprepared and unfilled roots was significantly higher than that of roots obturated with Gutta-percha/AH plus (SMD = - 0.69, 95% CI = - 1.34 to - 0.04, p = 0.04) or the Resilon system (SMD = - 0.54, 95% CI = - 1.07 to - 0.00, p = 0.05). The differences in fracture resistance between the roots filled with Gutta-percha/AH plus and the prepared unfilled root canals was not significant (SMD = 0.59, 95% CI = - 0.02 to 1.21, p = 0.06). Roots obturated with Resilon had higher fracture resistance than instrumented unfilled roots (SMD = 0.83, 95% CI = 0.44 to 1.22, p < 0.0001) or roots filled with Gutta-percha/AH plus (SMD = 0.62, 95% CI = 0.01 to 1.23, p = 0.05). CONCLUSIONS: The present study suggests that filling with Gutta-percha/AH plus dose not reinforce endodontically treated roots, whereas obturating with the Resilon system can increase vertical root fracture resistance of prepared roots. As this meta-analysis was based on in vitro studies, it should be careful to extrapolate its conclusion to the clinical context.

Genotype-Phenotype Correlation in NF1: Evidence for a More Severe Phenotype Associated with Missense Mutations Affecting NF1 Codons 844-848.

Neurofibromatosis type 1 (NF1), a common genetic disorder with a birth incidence of 1:2,000-3,000, is characterized by a highly variable clinical presentation. To date, only two clinically relevant intragenic genotype-phenotype correlations have been reported for NF1 missense mutations affecting p.Arg1809 and a single amino acid deletion p.Met922del. Both variants predispose to a distinct mild NF1 phenotype with neither externally visible cutaneous/plexiform neurofibromas nor other tumors. Here, we report 162 individuals (129 unrelated probands and 33 affected relatives) heterozygous for a constitutional missense mutation affecting one of five neighboring NF1 codons-Leu844, Cys845, Ala846, Leu847, and Gly848-located in the cysteine-serine-rich domain (CSRD). Collectively, these recurrent missense mutations affect ∼0.8% of unrelated NF1 mutation-positive probands in the University of Alabama at Birmingham (UAB) cohort. Major superficial plexiform neurofibromas and symptomatic spinal neurofibromas were more prevalent in these individuals compared with classic NF1-affected cohorts (both p < 0.0001). Nearly half of the individuals had symptomatic or asymptomatic optic pathway gliomas and/or skeletal abnormalities. Additionally, variants in this region seem to confer a high predisposition to develop malignancies compared with the general NF1-affected population (p = 0.0061). Our results demonstrate that these NF1 missense mutations, although located outside the GAP-related domain, may be an important risk factor for a severe presentation. A genotype-phenotype correlation at the NF1 region 844-848 exists and will be valuable in the management and genetic counseling of a significant number of individuals.

Effective Attenuation of Adenosine A1R Signaling by Neurabin Requires Oligomerization of Neurabin.

The adenosine A1 receptor (A1R) is a key mediator of the neuroprotective effect by endogenous adenosine. Yet targeting this receptor for neuroprotection is challenging due to its broad expression throughout the body. A mechanistic understanding of the regulation of A1R signaling is necessary for the future design of therapeutic agents that can selectively enhance A1R-mediated responses in the nervous system. In this study, we demonstrate that A1R activation leads to a sustained localization of regulator of G protein signaling 4 (RGS4) at the plasma membrane, a process that requires neurabin (a neural tissue-specific protein). A1R and RGS4 interact with the overlapping regions of neurabin. In addition, neurabin domains required for oligomerization are essential for formation of the A1R/neurabin/RGS4 ternary complex, as well as for stable localization of RGS4 at the plasma membrane and attenuation of A1R signaling. Thus, A1R and RGS4 each likely interact with one neurabin molecule in a neurabin homo-oligomer to form a ternary complex, representing a novel mode of regulation of G protein-coupled receptor signaling by scaffolding proteins. Our mechanistic analysis of neurabin-mediated regulation of A1R signaling in this study will be valuable for the future design of therapeutic agents that can selectively enhance A1R-mediated responses in the nervous system.