Biallelic variants in RBM42 cause a multisystem disorder with neurological, facial, cardiac, and musculoskeletal involvement.

Here, we report a previously unrecognized syndromic neurodevelopmental disorder associated with biallelic loss-of-function variants in the RBM42 gene. The patient is a 2-year-old female with severe central nervous system (CNS) abnormalities, hypotonia, hearing loss, congenital heart defects, and dysmorphic facial features. Familial whole-exome sequencing (WES) reveals that the patient has two compound heterozygous variants, c.304C>T (p.R102*) and c.1312G>A (p.A438T), in the RBM42 gene which encodes an integral component of splicing complex in the RNA-binding motif protein family. The p.A438T variant is in the RRM domain which impairs RBM42 protein stability in vivo. Additionally, p.A438T disrupts the interaction of RBM42 with hnRNP K, which is the causative gene for Au-Kline syndrome with overlapping disease characteristics seen in the index patient. The human R102* or A438T mutant protein failed to fully rescue the growth defects of RBM42 ortholog knockout ΔFgRbp1 in Fusarium while it was rescued by the wild-type (WT) human RBM42. A mouse model carrying Rbm42 compound heterozygous variants, c.280C>T (p.Q94*) and c.1306_1308delinsACA (p.A436T), demonstrated gross fetal developmental defects and most of the double mutant animals died by E13.5. RNA-seq data confirmed that Rbm42 was involved in neurological and myocardial functions with an essential role in alternative splicing (AS). Overall, we present clinical, genetic, and functional data to demonstrate that defects in RBM42 constitute the underlying etiology of a new neurodevelopmental disease which links the dysregulation of global AS to abnormal embryonic development.

Noninvasive Evaluation of Fetal Zygosity in Twin Pregnancies Involving a Binary Analysis of Single-Nucleotide Polymorphisms.

Twin pregnancy constitutes significant risks for maternal and fetal health, which is usually detected by ultrasound examination at early gestation. However, the imaging-based approach may not accurately identify all twins confounded by practical or clinical variables. The analysis of fetal cell-free DNA in noninvasive prenatal screening assays can completement the ultrasound method for twin detection, which differentiates fraternal or identical twins based on their distinct genotypes. Here, a new noninvasive prenatal screening employing high-coverage next-generation sequencing for targeted nucleotide polymorphisms was developed for detection of zygosity and determination of fetal fraction in twin pregnancies. This method utilizes a binary analysis of both the number and allelic fraction of fetus-specific single-nucleotide polymorphisms to infer the zygosity. In 323 samples collected from 215 singleton, 90 dizygotic, and 18 monozygotic twin pregnancies, all 90 dizygotic twins were correctly detected, with a 100% sensitivity and a 100% specificity. In addition, this method can detect complex pregnancies, such as egg donors, contamination, and twins with complete hydatidiform mole. The fetus-specific fetal fraction change was monitored in nine dizygotic twin pregnancies, which demonstrated highly variable dynamics of fetal cell-free DNA turnover up to 7 weeks after twin reduction. Overall, this study provides a new noninvasive prenatal screening strategy for the accurate identification of twin zygosity and quantification of fetal fraction, which has important clinical implications for the management of twin pregnancies.

Genetic deconvolution of fetal and maternal cell-free DNA in maternal plasma enables next-generation non-invasive prenatal screening.

Current non-invasive prenatal screening (NIPS) analyzes circulating fetal cell-free DNA (cfDNA) in maternal peripheral blood for selected aneuploidies or microdeletion/duplication syndromes. Many genetic disorders are refractory to NIPS largely because the maternal genetic material constitutes most of the total cfDNA present in the maternal plasma, which hinders the detection of fetus-specific genetic variants. Here, we developed an innovative sequencing method, termed coordinative allele-aware target enrichment sequencing (COATE-seq), followed by multidimensional genomic analyses of sequencing read depth, allelic fraction, and linked single nucleotide polymorphisms, to accurately separate the fetal genome from the maternal background. Analytical confounders including multiple gestations, maternal copy number variations, and absence of heterozygosity were successfully recognized and precluded for fetal variant analyses. In addition, fetus-specific genomic characteristics, including the cfDNA fragment length, meiotic error origins, meiotic recombination, and recombination breakpoints were identified which reinforced the fetal variant assessment. In 1129 qualified pregnancies tested, 54 fetal aneuploidies, 8 microdeletions/microduplications, and 8 monogenic variants were detected with 100% sensitivity and 99.3% specificity. Using the comprehensive cfDNA genomic analysis tools developed, we found that 60.3% of aneuploidy samples had aberrant meiotic recombination providing important insights into the mechanism underlying meiotic nondisjunctions. Altogether, we show that the genetic deconvolution of the fetal and maternal cfDNA enables thorough and accurate delineation of fetal genome which paves the way for the next-generation prenatal screening of essentially all types of human genetic disorders.

Low-level germline mosaicism of a novel SMARCA2 missense variant: Expanding the phenotypic spectrum and mode of genetic transmission.

BACKGROUND: Nicolaides-Baraitser syndrome (NCBRS) is a severe neurodevelopmental disorder with multiple abnormalities. To date, all pathogenic variants in SMARCA2 causing NCBRS are de novo and most are missense variants located in the ATPase domain of SMARCA2 protein. METHODS: In this study, a familial trio whole-exome sequencing was performed on the proband presenting with intellectual disability, early-onset epilepsy, and autistic features. A novel missense variant c.553C>G (p.Gln185Glu) in SMARCA2 was identified, which is located in the QLQ domain. The same variant was subsequently also found in the mother's ongoing pregnancy. Samples from accessible tissues such as saliva and sperm other than blood were collected from the parents, and the detection of the target variant was performed by amplicon-based deep sequencing. RESULTS: Low-level mosaicism of the target variant c.553C>G (p.Gln185Glu) was detected in the father's sperm with allele fraction of 2.8% by amplicon-based deep sequencing, which was not detected in either parents' blood or saliva specimens. Heterozygosity of this variant was confirmed in the proband. CONCLUSION: This is the first report of paternal germline mosaicism for a SMARCA2 disease-causing variant. In addition, the missense variant c.553C>G (p.Gln185Glu) in the QLQ domain causes mainly neurological and developmental phenotypes with unremarkable characteristic facial features and limb abnormalities. Our findings expand the phenotypic spectrum and mode of genetic transmission associated with the SMARCA2 variants.

Vitamin B12 modulates Parkinson's disease LRRK2 kinase activity through allosteric regulation and confers neuroprotection.

Missense mutations in Leucine-Rich Repeat Kinase 2 (LRRK2) cause the majority of familial and some sporadic forms of Parkinson's disease (PD). The hyperactivity of LRRK2 kinase induced by the pathogenic mutations underlies neurotoxicity, promoting the development of LRRK2 kinase inhibitors as therapeutics. Many potent and specific small-molecule LRRK2 inhibitors have been reported with promise. However, nearly all inhibitors are ATP competitive-some with unwanted side effects and unclear clinical outcome-alternative types of LRRK2 inhibitors are lacking. Herein we identify 5'-deoxyadenosylcobalamin (AdoCbl), a physiological form of the essential micronutrient vitamin B12 as a mixed-type allosteric inhibitor of LRRK2 kinase activity. Multiple assays show that AdoCbl directly binds LRRK2, leading to the alterations of protein conformation and ATP binding in LRRK2. STD-NMR analysis of a LRRK2 homologous kinase reveals the contact sites in AdoCbl that interface with the kinase domain. Furthermore, we provide evidence that AdoCbl modulates LRRK2 activity through disrupting LRRK2 dimerization. Treatment with AdoCbl inhibits LRRK2 kinase activity in cultured cells and brain tissue, and prevents neurotoxicity in cultured primary rodent neurons as well as in transgenic C. elegans and D. melanogaster expressing LRRK2 disease variants. Finally, AdoCbl alleviates deficits in dopamine release sustainability caused by LRRK2 disease variants in mouse models. Our study uncovers vitamin B12 as a novel class of LRRK2 kinase modulator with a distinct mechanism, which can be harnessed to develop new LRRK2-based PD therapeutics in the future.

Enhancing depression mechanisms in midbrain dopamine neurons achieves homeostatic resilience.

Typical therapies try to reverse pathogenic mechanisms. Here, we describe treatment effects achieved by enhancing depression-causing mechanisms in ventral tegmental area (VTA) dopamine (DA) neurons. In a social defeat stress model of depression, depressed (susceptible) mice display hyperactivity of VTA DA neurons, caused by an up-regulated hyperpolarization-activated current (I(h)). Mice resilient to social defeat stress, however, exhibit stable normal firing of these neurons. Unexpectedly, resilient mice had an even larger I(h), which was observed in parallel with increased potassium (K(+)) channel currents. Experimentally further enhancing Ih or optogenetically increasing the hyperactivity of VTA DA neurons in susceptible mice completely reversed depression-related behaviors, an antidepressant effect achieved through resilience-like, projection-specific homeostatic plasticity. These results indicate a potential therapeutic path of promoting natural resilience for depression treatment.

Imperfect interface of Beclin1 coiled-coil domain regulates homodimer and heterodimer formation with Atg14L and UVRAG.

Beclin 1 is a core component of the Class III Phosphatidylinositol 3-Kinase VPS34 complex. The coiled coil domain of Beclin 1 serves as an interaction platform for assembly of distinct Atg14L- and UVRAG-containing complexes to modulate VPS34 activity. Here we report the crystal structure of the coiled coil domain that forms an antiparallel dimer and is rendered metastable by a series of 'imperfect' a-d' pairings at its coiled coil interface. Atg14L and UVRAG promote the transition of metastable homodimeric Beclin 1 to heterodimeric Beclin1-Atg14L/UVRAG assembly. Beclin 1 mutants with their 'imperfect' a-d' pairings modified to enhance self-interaction, show distinctively altered interactions with Atg14L or UVRAG. These results suggest that specific utilization of the dimer interface and modulation of the homodimer-heterodimer transition by Beclin 1-interacting partners may underlie the molecular mechanism that controls the formation of various Beclin1-VPS34 subcomplexes to exert their effect on an array of VPS34-related activities, including autophagy.

Phosphorylation-dependent 14-3-3 binding to LRRK2 is impaired by common mutations of familial Parkinson's disease.

BACKGROUND: Recent studies show that mutations in Leucine Rich Repeat Kinase 2 (LRRK2) are the cause of the most common inherited and some sporadic forms of Parkinson's disease (PD). The molecular mechanism underlying the pathogenic role of LRRK2 mutations in PD remains unknown. METHODOLOGY/PRINCIPAL FINDINGS: Using affinity purification and mass spectrometric analysis, we investigated phosphorylation sites and binding proteins of LRRK2 purified from mouse brain. We identified multiple phosphorylation sites at N-terminus of LRRK2 including S910, S912, S935 and S973. Focusing on the high stoichiometry S935 phosphorylation site, we developed an anti-pS935 specific antibody and showed that LRRK2 is constitutively phosphorylated at S935 in various tissues (including brain) and at different ages in mice. We find that 14-3-3 proteins (especially isoforms γ and η) bind LRRK2 and this binding depends on phosphorylation of S935. The binding of 14-3-3, with little effect on dimer formation of LRRK2, confers protection of the phosphorylation status of S935. Furthermore, we show that protein kinase A (PKA), but not LRRK2 kinase itself, can cause the phosphorylation of LRRK2 at S935 in vitro and in cell culture, suggesting that PKA is a potential upstream kinase that regulates LRRK2 function. Finally, our study indicates that the common PD-related mutations of LRRK2, R1441G, Y1699C and G2019S, decrease homeostatic phosphorylation levels of S935 and impair 14-3-3 binding of LRRK2. CONCLUSIONS/SIGNIFICANCE: LRRK2 is extensively phosphorylated in vivo, and the phosphorylation of specific sites (e.g. S935) determines 14-3-3 binding of LRRK2. We propose that 14-3-3 is an important regulator of LRRK2-mediated cellular functions. Our study suggests that PKA, a cAMP-dependent kinase involved in regulating dopamine physiology, is a potential upstream kinase that phosphorylates LRRK2 at S935. Furthermore, the reduction of phosphorylation/14-3-3 binding of LRRK2 due to the common familial PD-related mutations provides novel insight into the pathogenic mechanism of LRRK2-linked PD.

Copper-1,10-phenanthroline-induced apoptosis in liver carcinoma Bel-7402 cells associates with copper overload, reactive oxygen species production, glutathione depletion and oxidative DNA damage.

The mechanism of cytotoxicity on liver carcinoma Bel-7402 cells induced by copper-1,10-phenanthroline, Cu(OP)2, has been studied. Cell viability and apoptotic rate were examined in cells treated with Cu(OP)2 or Cu2+ alone. It was found that the apoptosis induced by Cu(OP)2 could not be induced by Cu2+ or OP alone in our experimental conditions. Total copper content in cells was measured by atomic absorption spectrophotometry, and the abnormal elevation of intracellular copper transported by lipophilic OP ligand may play the role of initial factor in the apoptosis, which caused subsequent redox state changes in cells. Intracellular levels of reactive oxygen species (ROS) were detected by fluorescent probe 2',7'-dichlorofluorescein diacetate (DCFH-DA). Reduced (GSH) and total glutathione (GSSG + GSH) were determined by High-performance liquid chromatography (HPLC) after derivatization, and the ratios of GSH/GSSG were subsequently calculated. The overproduction of ROS and the decreased GSH/GSSG ratio were observed in cells which represented the occurrence of oxidative stress in the apoptosis. Oxidative DNA damage was also found in cells treated with Cu(OP)2 in the early stage of the apoptosis, and it suggests that the activation of DNA repair system may be involved in the pathway of the apoptosis induced by Cu(OP)2.

Unfolding features of bovine testicular hyaluronidase studied by fluorescence spectroscopy and fourier transformed infrared spectroscopy.

Chemical unfolding of bovine testicular hyaluronidase (HAase) has been studied by fluorescence spectroscopy and Fourier transformed infrared spectroscopy (FTIR). Thermodynamic parameters were determined for unfolding HAase from changes in the intrinsic fluorescence emission intensity and the formations of several possible unfolding intermediates have been identified. This was further confirmed by representation of fluorescence data in terms of 'phase diagram'. The secondary structures of HAase have been assigned and semiquantitatively estimated from the FTIR. The occurrence of conformational change during chemical unfolding as judged by fluorescence and FTIR spectroscopy indicated that the unfolding of HAase may not follow the typical two-state model.