Function Of Domain Research Articles

FGD1-related Aarskog–Scott syndrome: Identification of four novel variations and a literature review of clinical and molecular aspects

Patients with Aarskog–Scott syndrome (AAS) have short stature, facial anomalies, skeletal deformities, and genitourinary malformations. FYVE, RhoGEF, and PH domain-containing 1 (FGD1) is the only known causative gene of AAS. However, the diagnosis of AAS remains difficult, and specific treatments are still absent. Patients suspected with AAS were recruited, and clinical information was collected. Genetic testing and functional analysis were carried out for the diagnosis. By literature review, we summarized the clinical and genetic characteristics of FGD1-related AAS and analyzed the genotype–phenotype correlation. Five patients were recruited, and four novel FGD1 variants were identified. The diagnosis of AAS was confirmed by genetic analysis and functional study. Three patients treated with growth hormone showed improved heights during the follow-up period. By literature review, clinical features of AAS patients with FGD1 variants were summarized. Regarding FGD1 variations, substitutions were the most common form, and among them, missense variants were the most frequent. Moreover, we found patients with drastic variants showed higher incidences of foot and genitourinary malformations. Missense variants in DH domain were related to a lower incidence of cryptorchidism. Conclusion: We reported four novel pathogenic FGD1 variations in AAS patients and confirmed the efficacy and safety of growth hormone treatment in FGD1-related AAS patients with growth hormone deficiency. Additionally, our literature review suggested the crucial role of DH domain in FGD1 function.What is Known:• Aarskog–Scott syndrome is a rare genetic disease, and the only known cause is the variant in FGD1 gene. The typical clinical manifestations of AAS include facial, skeletal, and urogenital deformities and short stature.What is New:• We reported four novel FGD1 variants and reported the treatment of growth hormone in FGD1-related AAS patients. Our genotype–phenotype correlation analysis suggested the crucial role of DH domain in FGD1 function.

The 6 month interim analysis of a randomized controlled trial assessing the quality of life in patients with breast cancer related lymphedema undergoing lymphaticovenous anastomosis vs. conservative therapy

Breast cancer related lymphedema (BCRL) is a chronic condition with a detrimental impact on psychosocial and physical well-being. Lymphaticovenous anastomosis has shown promising results in alleviating physical symptoms and increasing quality of life in patients with BCRL. The aim of the study is to evaluate the effect on health related quality of life (HrQol) after LVA surgery versus conservative treatment in patients with BCRL. The study is a prospective, multicenter randomized controlled trial. Adult women with unilateral BCRL, with early stage lymphedema and viable lymphatic vessels were included. The primary outcome measure was HrQol measured by the lymphedema functioning disability and health (Lymph-ICF) questionnaire. The secondary outcomes were volume difference measured by the water displacement method; the Upper Extremity Lymphedema (UEL) index; and daily use of the compression garments after 3 and 6 months. For this interim analysis 46 patients per group were included. There was a significant improvement in the domains in physical and mental function in the Lymph-ICF questionnaire in the LVA group after 6 months, (− 16.46 ± 18.5, p < 0.05, − 10.12 ± 29.5, p < 0.05 respectively). However, there was no statistical difference in the total score of the Lymph-ICF after 6 months in both groups (LVA-group; − 8.57 ± 22.6, p > 0.05, CDT-group; − 2.65 ± 18.2, p < 0.05). Furthermore, there was no significant volume reduction in both groups (LVA-group: 20.04 ± 196.40, p = 0.497, CDT: 33.98 ± 189.87, p = 0.236). In the LVA group, 41% partially of completely stopped wearing the compression garments after six months whereas in the CDT group 0% discontinued to use of compression garments. LVA resulted in improvement of the domains physical and mental function of the Lymph-ICF. Limb volume did not significantly improve after 6 months. However, around 42% could completely or partially stopped with the use of compression garments in the LVA group. The current results are promising, however longer follow up is required to assess long term effect of LVA for secondary lymphedema. Clinical Trial Registration: NCT02790021 registered on 03/06/2016

Using High-Throughput Measurements to Identify Principles of Transcriptional and Epigenetic Regulators.

To achieve exquisite control over the epigenome, we need a better predictive understanding of how transcription factors, chromatin regulators, and their individual domain's function, both as modular parts and as full proteins. Transcriptional effector domains are one class of protein domains that regulate transcription and chromatin. These effector domains either repress or activate gene expression by interacting with chromatin-modifying enzymes, transcriptional cofactors, and/or general transcriptional machinery. Here, we discuss important design considerations for high-throughput investigations of effector domains, recent advances in discovering new domains in human cells and testing how domain function depends on amino acid sequence. For every effector domain, we would like to know the following: What role does the cell type, signaling state, and targeted context have on activation, silencing, and epigenetic memory? Large-scale measurements of transcriptional activities can help systematically answer these questions and identify general rules for how all these parameters affect effector domain activities. Last, we discuss what steps need to be taken to turn a newly discovered effector domain into a robust, precise epigenome editor. With more carefully considered high-throughput investigations, soon we will have better predictive control over the epigenome.

A novel leukocyte adhesion deficiency type III mutation manifests functional importance of the compact FERM domain in kindlin-3

BackgroundLeukocyte adhesion deficiency III (LAD-III) is a rare autosomal recessive syndrome characterized by functional deficiencies of platelets and leukocytes that occurs due to mutations in the FERMT3 gene encoding kindlin-3. Kindlin-3 is a FERM domain–containing adaptor protein that is essential in integrin activation. We have previously demonstrated that the FERM domain of kindlin-3 is structurally compact and plays an important role in supporting integrin activation in a mouse model. The impact of destabilizing the compact FERM domain in kindlin-3 on the development of LAD-III in humans remains uncertain. ObjectivesTo use primary cells from a patient with LAD-III to validate the role of the compact FERM domain in kindlin-3 function in platelets and leukocytes. MethodsThe patient is a 4-year-old girl who since infancy has displayed clinical features of LAD-III. Patient platelets and leukocytes were functionally analyzed, and structural analysis of the kindlin-3 variant was conducted. ResultsWe identified a novel homozygous missense mutation in the FERMT3 (c.412G>A, p.E138K) FERM domain. Substantially reduced levels of kindlin-3 were detected in the proband’s platelets and leukocytes. Functional evaluation verified that integrin αIIbβ3-mediated platelet activation, spreading, and aggregation and β2-integrin–mediated neutrophil adhesion and spreading were significantly compromised. Structural analysis revealed that this newly identified E138K substitution in kindlin-3 destabilizes the compacted FERM domain, resulting in poor expression of kindlin-3 in blood cells and subsequent LAD-III. ConclusionWe have identified a novel missense mutation and verified the functional significance of the compact kindlin-3 FERM domain in supporting integrin functions in platelets and leukocytes.

Transcriptional regulation by the NSL complex enables diversification of IFT functions in ciliated versus nonciliated cells.

Members of the NSL histone acetyltransferase complex are involved in multiorgan developmental syndromes. While the NSL complex is known for its importance in early development, its role in fully differentiated cells remains enigmatic. Using a kidney-specific model, we discovered that deletion of NSL complex members KANSL2 or KANSL3 in postmitotic podocytes led to catastrophic kidney dysfunction. Systematic comparison of two primary differentiated cell types reveals the NSL complex as a master regulator of intraciliary transport genes in both dividing and nondividing cells. NSL complex ablation led to loss of cilia and impaired sonic hedgehog pathway in ciliated fibroblasts. By contrast, nonciliated podocytes responded with altered microtubule dynamics and obliterated podocyte functions. Finally, overexpression of wild-type but not a double zinc finger (ZF-ZF) domain mutant of KANSL2 rescued the transcriptional defects, revealing a critical function of this domain in NSL complex assembly and function. Thus, the NSL complex exhibits bifurcation of functions to enable diversity of specialized outcomes in differentiated cells.

0912 Striatal atrophy and clinical correlates in REM sleep behavior disorder

Abstract Introduction Idiopathic/isolated REM sleep behavior disorder (iRBD) is regarded as the most specific prodromal stage of ⍺-synucleinopathies including Parkinson’s disease (PD). Striatal dopamine dysfunction is a pathological hallmark in PD, which has been similarly reported in iRBD as a sensitive marker for detecting early ⍺-synucleinopathies. In contrary, although there is a broad consensus that patients with PD have striatal volumes reduction, studies in iRBD are relatively scarce and inconsistent. In this study, we aim to investigate the difference of striatal nuclei volumes between iRBD and controls and their correlation with clinical presentations. Methods Altogether 105 patients (mean age=68.19, male 76.2%) with polysomnography-confirmed iRBD and 85 age and sex comparable controls (mean age=66.74, male 64.7%) underwent 3-tesla MRI and clinical assessments. FreeSurfer automatic segmentation pipelines measured the size of bilateral striatal nuclei including caudate, putamen, pallidum and accumbens area. The comparisons of the volume of individual striatal nucleus were made between iRBD and controls with age, sex, handness, and total intracranial volume as covariates. Correlations were performed to investigate the association between striatal nuclei volume and clinical domains in motor (Unified Parkinson’s Disease Rating Scale, Part III), cognitive (Montreal Cognitive Assessment, MoCA), visual (pareidolia), autonomic function (Scales for Outcomes in Parkinson’s Disease - Autonomic Dysfunction, SCOPA-AUT) and mood symptoms (Hospital Anxiety and Depression Scale). Results There were smaller volumes in the bilateral putamen (t=-2.13/-2.32, p=0.03/0.02, left/right side respectively) and accumbens area (t=-2.58/-2.07, p=0.01/0.04, left/right side respectively) in iRBD comparing to controls. In addition, MoCA score was significantly positively correlated with the volumes of bilateral putamen (r=0.212/0.213, p=0.032/0.033), while pareidolia score was significantly negatively correlated with volumes of bilateral putamen (r=-0.205/-0.233, p=0.039/0.018,) and accumbens area (r=-0.249/-0.279, p=0.012/0.004, left/right respectively). The SCOPA-AUT score was significantly negatively correlated with volumes of the left putamen (r=-0.286, p=0.014) and the left accumbens area (r=-0.245, p=0.037). No other correlations with mood and motor features were found. Conclusion In this large case-control study, PD-like striatal atrophy was already present in patients with iRBD, which were significantly associated with worse cognitive performance, visual and autonomic dysfunction. Support (if any) This study was supported by Hong Kong Health and Medical Research Fund, 08191416.

A Twist-Box domain of the C. elegans Twist homolog, HLH-8, plays a complex role in transcriptional regulation.

TWIST1 is a basic helix-loop-helix (bHLH) transcription factor in humans that functions in mesoderm differentiation. TWIST1 primarily regulates genes as a transcriptional repressor often through TWIST-Box domain-mediated protein-protein interactions. The TWIST-Box also can function as an activation domain requiring 3 conserved, equidistant amino acids (LXXXFXXXR). Autosomal dominant mutations in TWIST1, including 2 reported in these conserved amino acids (F187L and R191M), lead to craniofacial defects in Saethre-Chotzen syndrome (SCS). Caenorhabditis elegans has a single TWIST1 homolog, HLH-8, that functions in the differentiation of the muscles responsible for egg laying and defecation. Null alleles in hlh-8 lead to severely egg-laying defective and constipated animals due to defects in the corresponding muscles. TWIST1 and HLH-8 share sequence identity in their bHLH regions; however, the domain responsible for the transcriptional activity of HLH-8 is unknown. Sequence alignment suggests that HLH-8 has a TWIST-Box LXXXFXXXR motif; however, its function also is unknown. CRISPR/Cas9 genome editing was utilized to generate a domain deletion and several missense mutations, including those analogous to SCS patients, in the 3 conserved HLH-8 amino acids to investigate their functional role. The TWIST-Box alleles did not phenocopy hlh-8 null mutants. The strongest phenotype detected was a retentive (Ret) phenotype with late-stage embryos in the hermaphrodite uterus. Further, GFP reporters of HLH-8 downstream target genes (arg-1::gfp and egl-15::gfp) revealed tissue-specific, target-specific, and allele-specific defects. Overall, the TWIST-Box in HLH-8 is partially required for the protein's transcriptional activity, and the conserved amino acids contribute unequally to the domain's function.

The SOCS1 KIR and SH2 domain are both required for suppression of cytokine signaling in vivo

Suppressor Of Cytokine Signaling (SOCS) 1 is a critical negative regulator of cytokine signaling and required to protect against an excessive inflammatory response. Genetic deletion of Socs1 results in unrestrained cytokine signaling and neonatal lethality, characterised by an inflammatory immune infiltrate in multiple organs. Overexpression and structural studies have suggested that the SOCS1 kinase inhibitory region (KIR) and Src homology 2 (SH2) domain are important for interaction with and inhibition of the receptor-associated JAK1, JAK2 and TYK2 tyrosine kinases, which initiate downstream signaling. To investigate the role of the KIR and SH2 domain in SOCS1 function, we independently mutated key conserved residues in each domain and analysed the impact on cytokine signaling, and the in vivo impact on SOCS1 function. Mutation of the SOCS1-KIR or SH2 domain had no impact on the integrity of the SOCS box complex, however, mutation within the phosphotyrosine binding pocket of the SOCS1-SH2 domain specifically disrupted SOCS1 interaction with phosphorylated JAK1. In contrast, mutation of the KIR did not affect the interaction with JAK1, but did prevent SOCS1 inhibition of JAK1 autophosphorylation. In human and mouse cell lines, both mutants impacted the ability of SOCS1 to restrain cytokine signaling, and crucially, Socs1-R105A and Socs1-F59A mice displayed a neonatal lethality and excessive inflammatory phenotype similar to Socs1-null mice. This study defines a critical and non-redundant role for both the KIR and SH2 domain in endogenous SOCS1 function.

Activity-based CRISPR scanning uncovers allostery in DNA methylation maintenance machinery.

Allostery enables dynamic control of protein function. A paradigmatic example is the tightly orchestrated process of DNA methylation maintenance. Despite the fundamental importance of allosteric sites, their identification remains highly challenging. Here, we perform CRISPR scanning on the essential maintenance methylation machinery-DNMT1 and its partner UHRF1-with the activity-based inhibitor decitabine to uncover allosteric mechanisms regulating DNMT1. In contrast to non-covalent DNMT1 inhibition, activity-based selection implicates numerous regions outside the catalytic domain in DNMT1 function. Through computational analyses, we identify putative mutational hotspots in DNMT1 distal from the active site that encompass mutations spanning a multi-domain autoinhibitory interface and the uncharacterized BAH2 domain. We biochemically characterize these mutations as gain-of-function, exhibiting increased DNMT1 activity. Extrapolating our analysis to UHRF1, we discern putative gain-of-function mutations in multiple domains, including key residues across the autoinhibitory TTD-PBR interface. Collectively, our study highlights the utility of activity-based CRISPR scanning for nominating candidate allosteric sites, and more broadly, introduces new analytical tools that further refine the CRISPR scanning framework.

Structural dynamics of the activation loop in the kinase domain of E.faecalis IreK.

IreK is a transmembrane Ser/Thr PASTA kinase found in Enterococcus faecalis, as well as most other Gram-positive bacteria, and plays a key role in resistance to cell wall-active antibiotics such as cephalosporins. IreK belongs to a family of bacterial transmembrane kinases that include multiple PASTA (Penicillin-and Ser/Thr-kinase Associated) domains in the extracellular segment and are ubiquitous in nearly all Gram-positive bacteria. PASTA kinases have conserved functions across Gram-positive bacteria involving monitoring and regulating processes associated with the cell wall. Enterococci are frequently found in the human gut and are a common cause of severe and difficult to treat hospital acquired infections, due to their intrinsic drug resistance. The underlying mechanisms of antibiotic resistance in enterococci are not well understood and are an excellent target for the creation of preventative treatments to eliminate the growth opportunity of resistant E. faecalis in the gut and transfer to other parts of the body. Based on the crystal structures of IreK kinase domain homologs, the activation loop structure is unknown and the phosphorylation sites within this loop are proposed to play a key regulatory role in IreK kinase activity. IreB has also been identified as one of the targets for phosphorylation by IreK. Here, continuous wave electron paramagnetic resonance (CW EPR) spectroscopy was applied to study the dynamics of the IreK activation loop, including the effects of phosphorylation and IreB binding on activation loop structure and dynamics. In addition, new functionally important non-phosphorylation sites in the activation loop that regulate IreK function in vivo were identified and characterized. This comprehensive investigation into the IreK activation loop structure and functional dynamics characterizes the role of this loop as a key regulatory domain in kinase function.

NLRP3 leucine-rich repeats control induced and spontaneous inflammasome activation in cryopyrin-associated periodic syndrome

The cryopyrin-associated periodic syndromes (CAPS) comprise a group of rare autoinflammatory diseases caused by gain-of-function mutations in the NLRP3 gene. NLRP3 contains a leucine-rich repeats (LRR) domain with a highly conserved exonic organization that is subjected to extensive alternative splicing. Aberrant NLRP3 inflammasome assembly in CAPS causes chronic inflammation; however, the mechanisms regulating inflammasome function remain unclear. We aimed to elucidate the mechanisms regulating NLRP3-mediated autoinflammation in human disease, characterizing the role of LRR in inflammasome activation. We analyzed sequence read archive data to characterize the pattern of NLRP3 splicing in human monocytes and investigated the role of each LRR-coding exon in inflammasome regulation in genetically modified U937 cells representing CAPS and healthy conditions. We detected a range of NLRP3 splice variants in human primary cells and monocytic cell lines, including 2 yet-undescribed splice variants. We observe that lipopolysaccharides affect the abundance of certain splice variants, suggesting that they may regulate NLRP3 activation by affecting alternative splicing. We showed that exons 4, 5, 7, and 9 are essential for inflammasome function, both in the context of wild-type NLRP3 activation by the agonist molecule nigericin and in a model of CAPS-mediated NLRP3 inflammasome assembly. Moreover, the SGT1-NLRP3 interaction is decreased in nonfunctional variants, suggesting that alternative splicing may regulate the recruitment of proteins that facilitate inflammasome assembly. These findings demonstrate the contribution of the LRR domain in inflammasome function and suggest that navigating LRR exon usage within NLRP3 is sufficient to dampen inflammasome assembly in CAPS.

Interaction between VPS13A and the XK scramblase is important for VPS13A function in humans.

VPS13 family proteins form conduits between the membranes of different organelles through which lipids are transferred. In humans, there are four VPS13 paralogs, and mutations in the genes encoding each of them are associated with different inherited disorders. VPS13 proteins contain multiple conserved domains. The Vps13 adaptor-binding (VAB) domain binds to adaptor proteins that recruit VPS13 to specific membrane contact sites. This work demonstrates the importance of a different domain in VPS13A function. The pleckstrin homology (PH) domain at the C-terminal region of VPS13A is required to form a complex with the XK scramblase and for the co-localization of VPS13A with XK within the cell. Alphafold modeling was used to predict an interaction surface between VPS13A and XK. Mutations in this region disrupt both complex formation and co-localization of the two proteins. Mutant VPS13A alleles found in patients with VPS13A disease truncate the PH domain. The phenotypic similarities between VPS13A disease and McLeod syndrome caused by mutations in VPS13A and XK, respectively, argue that loss of the VPS13A-XK complex is the basis of both diseases.

Clinical, functional, and patient-reported outcome of traumatic knee dislocations: a retrospective cohort study of 75 patients with 6.5-year follow-up.

At present, limited knowledge regarding clinical, functional, and patient-reported outcomes at mid- and long-terms after surgical treatment of traumatic knee dislocations is available. This study aimed to investigate the mid-term recovery regarding clinical, functional, and patient-reported outcomes in patients following knee dislocation with associated multi-ligament injuries. The study design was a cross-sectional cohort study. Data were collected by retrospective chart review, clinical examination, and interview of patients. All patients treated surgically following a knee dislocation between January 2000 and December 2011 were included. The surgical technique was up to the decision of the individual surgeon. The main outcome was the Lysholm knee score. Secondary outcomes consist of clinical knee examination, functional performance test, pain, and patient-reported outcome across several domains in function, sport, pain, and quality of life. Seventy-five patients (66.3%) accepted the invitation to participate. The mean age at the time of knee dislocation was 33.5years, with a range of 16-65years of age. The mean follow-up time was 78months (R: 17-147). 75% of patient a Schenck's type 1 lesion and 23% a type 3. The median Lysholm knee score was 83 (R: 18-100). The mean KOOS for the five subscales were pain 84.5 (95% CI 80.5-88.5), symptoms 75.1 (95% CI 70.7-79.4), ADL 87.0 (95% CI 83.1-90.9), sport 59.9 (95% CI 53.3-66.4), and QOL 71.3 (95% CI 67.0-75.6). The mean Tegner activity level was 5.1 (95% CI 4.5-5.7). The median single assessment numeric evaluation (SANE) was 93 (R: 0-100). The pain intensity score for pain (VAS) during activity was reported with a mean of 2.7 (95% CI 2.1-3.3). The objective IKDC examination showed 76% of patients grouped by Grade A (normal knee function) or Grade B (nearly normal). With a mean follow-up of 6.5years, combined repair and reconstruction surgery following a knee dislocation shows good to excellent patient-reported outcome and more than 75% of patients experiencing normal knee functioned evaluated by the IKDC score.

A conformational transition of the D′D3 domain primes von Willebrand factor for multimerization

AbstractVon Willebrand factor (VWF) is a multimeric plasma glycoprotein that is critically involved in hemostasis. Biosynthesis of long VWF concatemers in the endoplasmic reticulum and the trans-Golgi is still not fully understood. We use the single-molecule force spectroscopy technique magnetic tweezers to analyze a previously hypothesized conformational change in the D′D3 domain crucial for VWF multimerization. We find that the interface formed by submodules C8-3, TIL3, and E3 wrapping around VWD3 can open and expose 2 buried cysteines, Cys1099 and Cys1142, that are vital for multimerization. By characterizing the conformational change at varying levels of force, we can quantify the kinetics of the transition and stability of the interface. We find a pronounced destabilization of the interface on lowering the pH from 7.4 to 6.2 and 5.5. This is consistent with initiation of the conformational change that enables VWF multimerization at the D′D3 domain by a decrease in pH in the trans-Golgi network and Weibel-Palade bodies. Furthermore, we find a stabilization of the interface in the presence of coagulation factor VIII, providing evidence for a previously hypothesized binding site in submodule C8-3. Our findings highlight the critical role of the D′D3 domain in VWF biosynthesis and function, and we anticipate our methodology to be applicable to study other, similar conformational changes in VWF and beyond.

Model for surface-dependent factor XII activation: the roles of factor XII heavy chain domains

AbstractFactor XII (FXII) is the zymogen of a plasma protease (FXIIa) that contributes to bradykinin generation by converting prekallikrein to the protease plasma kallikrein (PKa). FXII conversion to FXIIa by autocatalysis or PKa-mediated cleavage is enhanced when the protein binds to negatively charged surfaces such as polymeric orthophosphate. FXII is composed of noncatalytic (heavy chain) and catalytic (light chain) regions. The heavy chain promotes FXII surface-binding and surface-dependent activation but restricts activation when FXII is not surface bound. From the N terminus, the heavy chain contains fibronectin type 2 (FN2), epidermal growth factor-1 (EGF1), fibronectin type 1 (FN1), EGF2, and kringle (KNG) domains and a proline-rich region. It shares this organization with its homolog, pro–hepatocyte growth factor activator (Pro-HGFA). To study the importance of heavy chain domains in FXII function, we prepared FXII with replacements of each domain with corresponding Pro-HGFA domains and tested them in activation and activity assays. EGF1 is required for surface-dependent FXII autoactivation and surface-dependent prekallikrein activation by FXIIa. KNG and FN2 are important for limiting FXII activation in the absence of a surface by a process that may require interactions between a lysine/arginine binding site on KNG and basic residues elsewhere on FXII. This interaction is disrupted by the lysine analog ε-aminocaproic acid. A model is proposed in which an ε-aminocaproic acid–sensitive interaction between the KNG and FN2 domains maintains FXII in a conformation that restricts activation. Upon binding to a surface through EGF1, the KNG/FN2-dependent mechanism is inactivated, exposing the FXII activation cleavage site.

Exosite binding modulates the specificity of the immunomodulatory enzyme ScpA, a C5a inactivating bacterial protease

The C5a peptidase from Streptococcus pyogenes (ScpA) is a highly specific enzyme with potential therapeutic value. ScpA is a good model for studying determinants of specificity in the multidomain immunomodulatory enzymes (IMEs), which comprise a large family of bacterial surface proteases. The surface exposed region of ScpA has 5 main domains which includes 3 C-terminal Fn3-like domains (Fn1, Fn2 and Fn3) (Kagawa et al. 2009). Progressive deletion of the Fn3-like domains from the C-ter resulted in loss of enzyme activity and showed an important role for the Fn2 domain in enzyme function. Functional investigation of specific acidic residues on the Fn2 domain identified 3 residues 30–50 Å from the catalytic site (D783, E864 and D889) which impacted to differing degrees on binding and on catalysis, supporting the presence of an exosite on the Fn2. In particular, residue D783 was observed to impact on both substrate binding affinity and the activity of ScpA. A double mutant cycle analysis showed energetic coupling between the targeted ScpA residues and residues in the core portion (residues 1–67) of the C5a substrate. The data supports the presence of a communication network between the active site and the exosite on Fn2. These findings provide a basis for rational engineering of this important enzyme family to enhance stability, activity and/or specificity.

The influence of known pathogenic gene mutation and ApoEε4 on neuropsychological evaluation and imaging markers in preclinical stage of familial Alzheimer’s disease

AbstractBackgroundIn the preclinical stage of familial Alzheimer's disease (FAD), there have been changes in multi‐dimensional indicators, such as pathology, neuropsychology and imaging. This study explored the effects of known pathogenic gene mutation and ApoE on neuropsychology and imaging markers before the appearance of FAD symptoms.Method102 cases of asymptomatic mutation non‐carriers (26 cases of ApoEε4) and 38 cases of asymptomatic mutation carriers (17 APP, 21 PS1) in FAD families were assessed by a series of neuropsychological scales, including mini mental state scale (MMSE), auditory verbal learning test (AVLT), etc. MRI images were collected. The volume of representative brain regions was obtained, including subregions of striatum, hippocampus, rostral middle frontal gyrus (rMFG), and posterior cingulate cortex (PCC). Diffusion indices and functional connectivity (FC) of neural pathways (striatum subregions to rMFG, hippocampus to PCC) were also obtained. The neuropsychological scores and imaging indexes were compared between groups, and the imaging indexes with group differences were further correlated with neuropsychological scores.ResultCompared with asymptomatic mutation non‐carriers, APP group showed a trend increase in delayed recall score in AVLT (P = 0.08), significantly increased cued recall score, and increased radial diffusivity (RD) of bilateral caudate‐rMFG pathway (fig 1, P's &lt; 0.05). In asymptomatic mutation non‐carriers, compared with APOEε4 non‐carriers, APOEε4 carriers showed increased right hippocampal volume, decreased fractional anisotropy (FA) of bilateral hippocampus‐PCC pathway, and increased FC of left caudate‐rMFG pathway (fig 2, P's &lt; 0.05). The RD of right caudate‐rMFG pathway in PS1 group was positively correlated with MMSE total score (r = 0.749). The FC of left caudate‐rMFG pathway was negatively correlated with MMSE total score (fig 3, r = ‐ 0.623, P’s &lt; 0.05).ConclusionThere are neuropsychological and imaging changes in the early stage of FAD, which are affected by the known pathogenic gene mutation and ApoEε4. APP mutation affected memory domain in cognitive function and the structural connectivity of fronto‐striatal pathway. ApoEε4 mainly affected the structural connectivity of hippocampus‐PCC pathway. The hippocampal volume and the functional connectivity of fronto‐striatal pathway increased compensatively. The overall cognitive function is differently related to the structural and functional connectivity of fronto‐striatal pathway in APP, PS1 and ApoEε4.

Healthy Diet for Healthy Aging.

Extending healthspan is a major public health challenge. Diet is one of the modifiable factors for preventing age-related diseases and preserving overall good health status during aging. Optimizing individuals’ intrinsic capacity, including domains in cognition, psychological, sensory function, vitality, and locomotion, has been proposed as a model of healthy aging by the World Health Organization. To better understand the relationships between a healthy diet and healthy aging, this review summarizes the recent epidemiologic and clinical data for dietary patterns that have been shown to play a role in (domains of) healthy aging. Regardless of priori or posteriori dietary patterns, it appears that dietary patterns centered on plant-based foods have a beneficial role in (domains of) healthy aging. Our review identified a knowledge gap in dietary patterns and multidimensional concepts of healthy aging. More epidemiological studies should consider intrinsic capacity as an outcome measure to further our understanding of a healthy diet and multidimensional concepts of healthy aging. When a sufficient number of epidemiological studies is available, evidence can be synthesized and clinical trials can be designed to evaluate a healthy diet as a strategy for healthy aging to further our progress in translating evidence to practice and promoting healthy aging.

Fetal Hemoglobin (HbF) Silencer BCL11A Acts through a Novel DNA-Motif in the Gamma-Globin Promoters, Simplifying the Model for Hemoglobin Switching

Fetal hemoglobin (HbF) level is under strong genetic control. Common genetic variation is associated with modest elevation in HbF levels in adults. Genome-wide association studies led to identification of the regulatory factor BCL11A, which is a major contributor to HbF silencing. Despite clear evidence for its involvement, how BCL11A acts to repress gamma-globin gene expression has remained unknown. Conflicting data exist in the literature with regard to whether BCL11A binds DNA directly and, if so, the sequence(s) it may recognize. Prior experiments from our laboratory suggested that BCL11A acts at a distance from the gamma-globin gene promoters, but failed to define with confidence sites of occupancy within the beta globin locus. To explore mechanisms by which BCL11A functions, we first devised a cellular assay for rescue of BCL11A-deficient erythroid cells with BCL11A cDNA constructs. This approach established that of the two longer BCL11A isoforms (L and XL), only XL (“eXtraLong”) is competent to repress globin expression in cells. XL differs from L by the presence of an alternatively spliced terminal exon encoding three closely spaced zinc-fingers (ZF4-6), implicating this domain in an essential function of the protein. Given that ZFs often mediate sequence-specific DNA-binding, we employed a Protein Binding Microarray platform (PBM) that allows for high-throughput determination of transcription factor binding specificities at high-throughput on an oligonucleotide matrix. We determined that recombinant ZF4-6, as well as BCL11A-XL, binds sequence-specifically to a novel consensus motif with a central core, TGACCA. Two biophysical methods confirmed recognition of this motif by recombinant ZF4-6 with an affinity of ~ 25nM. The motif is present in promoters of all fetal or embryonic-expressed globin genes in human and mouse, and in two copies in each human gamma-globin gene promoter (-119 to -113 and -92 to -86). The motif is altered by single base substitutions in rare HPFH alleles (-117 and -114) and by a microdeletion HPFH (-114 to -102). HPFH-associated single base substitutions greatly impair ZF4-6 binding, therefore strongly implicating a direct role for BCL11A at the gamma-globin gene promoters. However, numerous attempts to localize endogenous BCL11A protein in chromatin of erythroid cells by conventional chromatin immunoprecipitation sequencing (ChIP-seq) proved unsuccessful. To determine if the gamma-globin gene promoters are indeed bound by BCL11A in vivo in erythroid cells, we adapted a newly described method for detection of protein occupancy in chromatin (CUT&RUN) at higher resolution and without crosslinking that is commonly used in ChIP-seq. With this method, we detected BCL11A occupancy in the gamma-globin gene promoters in adult erythroid cells, including CD34 cells, and moreover observed that the distal one of the repeated motifs is the predominant binding site in vivo . Taken together, these experiments provide persuasive evidence that BCL11A acts locally at the gamma-globin gene promoters to repress transcription and that classical HPFH alleles in the (-117 to -114) region lead to increased HbF due to failure of BCL11A binding. These findings are consistent with a revised model of HbF silencing in which BCL11A acts through direct gamma-globin promoter binding, but do not exclude additional architectural or long-distance roles of BCL11A in HbF silencing. The critical role of the BCL11A DNA-binding motif in mediating HbF repression further supports the rationale for use of genome editing to target this region as therapy of the hemoglobinopathies. DisclosuresOrkin:Bioverativ: Consultancy; Epizyme Inc.: Consultancy.

MYH7-related disorders in two Bulgarian families: Novel variants in the same region associated with different clinical manifestation and disease penetrance

Pathogenic variants in MYH7 cause a wide range of cardiac and skeletal muscle diseases with childhood or adult onset. These include dilated and/or hypertrophic cardiomyopathy, left ventricular non-compaction cardiomyopathy, congenital myopathies with multi-minicores and myofiber type disproportion, myosin storage myopathy, Laing distal myopathy and others (scapulo-peroneal or limb-girdle muscle forms). Here we report the results from molecular genetic analyses (NGS and Sanger sequencing) of 4 patients in two families with variable neuromuscular phenotypes with or without cardiac involvement. Interestingly, variants in MYH7 gene appeared to be the cause in all the cases. A novel nonsense variant c.5746C>T, p.(Gln1916Ter) was found in the patient in Family 1 who deceased at the age of 2 years 4 months with the clinical diagnosis of dilated cardiomyopathy, whose father died before the age of 40 years, due to cardiac failure with clinical diagnosis of suspected limb-girdle muscular dystrophy. A splice acceptor variant c.5560–2A>C in MYH7 was detected in the second proband and her sister, with late onset distal myopathy without cardiac involvement. These different phenotypes (muscular involvement with severe cardiomyopathy and pure late onset neuromuscular phenotype without heart involvement) may result from novel MYH7 variants, which most probably impact the LMM (light meromyosin) domain's function of the mature protein.