N-terminal Variants Research Articles

Downstream Alternate Start Site Allows N-Terminal Nonsense Variants to Escape NMD and Results in Functional Recovery by Readthrough and Modulator Combination.

Genetic variants that introduce premature termination codons (PTCs) have remained difficult to therapeutically target due to lack of protein product. Nonsense mediated mRNA decay (NMD) targets PTC-bearing transcripts to reduce the potentially damaging effects of truncated proteins. Readthrough compounds have been tested on PTC-generating variants in attempt to permit translation through a premature stop. However, readthrough compounds have not proved efficacious in a clinical setting due to lack of stable mRNA. Here, we investigate N-terminal variants in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which have been shown to escape NMD, potentially through a mechanism of alternative translation initiation at downstream AUG codons. We hypothesized that N-terminal variants in CFTR that evade NMD will produce stable transcript, allowing CFTR function to be restored by a combination of readthrough and protein modulator therapy. We investigate this using two cell line models expressing CFTR-expression minigenes (EMG; HEK293s and CFBEs) and primary human nasal epithelial (NE) cells, and we test readthrough compounds G418 and ELX-02 in combination with CFTR protein modulators. HEK293 cells expressing the variants E60X and L88X generate CFTR-specific core glycosylated products that are consistent with downstream translation initiation. Mutation of downstream methionines at codons 150 and 152 does not result in changes in CFTR protein processing in cells expressing L88X-CFTR-EMG. However, mutation of methionine at 265 results in loss of detectable CFTR protein in cells expressing E60X, L88X, and Y122X CFTR-EMGs, indicating that downstream translation initiation is occurring at the AUG codon at position M265. In HEK293 stable cells harboring L88X, treatment with readthrough compounds alone allows for formation of full-length, but misfolded CFTR protein. Upon addition of protein modulators in combination with readthrough, we observe formation of mature, complex-glycosylated CFTR. In CFBE and NE cells, addition of readthrough ELX-02 and modulator therapy results in substantial recovery of CFTR function. Our work indicates that N-terminal variants generate stable CFTR transcript due to translation initiation at a downstream AUG codon. Thus, individuals with CF bearing 5′ nonsense variants that evade NMD are ideal candidates for treatment with clinically safe readthrough compounds and modulator therapy.

Molecular and clinical descriptions of patients with GABAA receptor gene variants (GABRA1, GABRB2, GABRB3, GABRG2): A cohort study, review of literature, and genotype-phenotype correlation.

γ-Aminobutyric acid (GABA)A -receptor subunit variants have recently been associated with neurodevelopmental disorders and/or epilepsy. The phenotype linked with each gene is becoming better known. Because of the common molecular structure and physiological role of these phenotypes, it seemed interesting to describe a putative phenotype associated with GABAA -receptor-related disorders as a whole and seek possible genotype-phenotype correlations. We collected clinical, electrophysiological, therapeutic, and molecular data from patients with GABAA -receptor subunit variants (GABRA1, GABRB2, GABRB3, and GABRG2) through a national French collaboration using the EPIGENE network and compared these data to the one already described in the literature. We gathered the reported patients in three epileptic phenotypes: 15 patients with fever-related epilepsy (40%), 11 with early developmental epileptic encephalopathy (30%), 10 with generalized epilepsy spectrum (27%), and 1 patient without seizures (3%). We did not find a specific phenotype for any gene, but we showed that the location of variants on the transmembrane (TM) segment was associated with a more severe phenotype, irrespective of the GABAA -receptor subunit gene, whereas N-terminal variants seemed to be related to milder phenotypes. GABAA -receptor subunit variants are associated with highly variable phenotypes despite their molecular and physiological proximity. None of the genes described here was associated with a specific phenotype. On the other hand, it appears that the location of the variant on the protein may be a marker of severity. Variant location may have important weight in the development of targeted therapeutics.

Functional antagonism between CagA and DLC1 in gastric cancer

Helicobacter (H.) pylori-induced gastritis is a risk factor for gastric cancer (GC). Deleted-in-liver-cancer-1 (DLC1/ARHGAP7) inhibits RHOA, a downstream mediator of virulence factor cytotoxin-A (CagA) signalling and driver of consensus-molecular-subtype-2 diffuse GC. DLC1 located to enterochromaffin-like and MIST1+ stem/chief cells in the stomach. DLC1+ cells were reduced in H. pylori gastritis and GC, and in mice infected with H. pylori. DLC1 positivity inversely correlated with tumour progression in patients. GC cells retained an N-terminal truncation variant DLC1v4 in contrast to full-length DLC1v1 in non-neoplastic tissues. H. pylori and CagA downregulated DLC1v1/4 promoter activities. DLC1v1/4 inhibited cell migration and counteracted CagA-driven stress phenotypes enforcing focal adhesion. CagA and DLC1 interacted via their N- and C-terminal domains, proposing that DLC1 protects against H. pylori by neutralising CagA. H. pylori-induced DLC1 loss is an early molecular event, which makes it a potential marker or target for subtype-aware cancer prevention or therapy.

Characterization of N-terminal RYR2 variants outside CPVT1 hotspot regions using patient iPSCs reveal pathogenesis and therapeutic potential.

SummaryCatecholaminergic polymorphic ventricular tachycardia (CPVT) is a cardiac channelopathy causing ventricular tachycardia following adrenergic stimulation. Pathogenic variants in RYR2-encoded ryanodine receptor 2 (RYR2) cause CPVT1 and cluster into domains I–IV, with the most N-terminal domain involving residues 77–466. Patient-specific induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) were generated for RYR2-F13L, -L14P, -R15P, and -R176Q variants. Isogenic control iPSCs were generated using CRISPR-Cas9/PiggyBac. Fluo-4 Ca2+ imaging assessed Ca2+ handling with/without isoproterenol (ISO), nadolol (Nad), and flecainide (Flec) treatment. CPVT1 iPSC-CMs displayed increased Ca2+ sparking and Ca2+ transient amplitude following ISO compared with control. Combined Nad treatment/ISO stimulation reduced Ca2+ amplitude and sparking in variant iPSC-CMs. Molecular dynamic simulations visualized the structural role of these variants. We provide the first functional evidence that these most proximal N-terminal localizing variants alter calcium handling similar to CPVT1. These variants are located at the N-terminal domain and the central domain interface and could destabilize the RYR2 channel promoting Ca2+ leak-triggered arrhythmias.

Inclusion of a dual signal sequence enhances the immunogenicity of a novel viral vectored vaccine against the capsular group B meningococcus

BackgroundDisease caused by the capsular group B meningococcus (MenB) is the leading cause of infectious death in UK infants. A novel adenovirus-based vaccine encoding the MenB factor H binding protein (fHbp) with an N-terminal dual signal sequence induces high titres of protective antibody after a single dose in mice. A panel of N-terminal signal sequence variants were created to assess the contribution of components of this sequence to transgene expression kinetics of the encoded antigen from mammalian cells and the resultant effect on immunogenicity of fHbp.ResultsThe full-length signal sequence (FL SS) resulted in superior early antigen expression compared with the panel of variants, as measured by flow cytometry and confocal imaging, and supported higher bactericidal antibody levels against the expressed antigen in mouse sera < 6 weeks post-immunisation than the licensed four component MenB vaccine. The FL SS also significantly increased antigen-specific T cell responses against other adenovirus-encoded bacterial antigens in mice.ConclusionsThese findings demonstrate that the FL SS enhances immunogenicity of the encoded antigen, supporting its inclusion in other viral vectored bacterial antigen transgenes.

An exceptional biallelic N-terminal frame shift mutation in ZMPSTE24 leads to non-lethal progeria due to possible utilization of a downstream alternative start codon

Biallelic mutations in ZMPSTE24 are known to be associated with autosomal recessive mandibuloacral dysplasia with type B lipodystrophy (MADB) and lethal restrictive dermopathy (RD), respectively. Disease manifestation is depending on the remaining enzyme activity of the mutated ZMPSTE24 protein. To date, complete loss of function has exclusively been reported in RD cases. In this study, we identified a novel N-terminal homozygous frameshift mutation (c.28_29insA) in a consanguineous family segregating with MADB. An in-depth analysis of the mutated sequence revealed, that the one base pair insertion creates a novel downstream in-frame start codon, which supposedly serves as an alternative translation initiation site (TIS). This possible rescue mechanism would explain the relatively mild clinical outcome in the studied individuals.Our findings demonstrate the necessity for careful interpretation of N-terminal variants potentially effecting translation initiation.

Mild neurological phenotype in a family carrying a novel N-terminal null GRIN2A variant

GRIN2A encodes for the 2A subunit of N-methyl-D-aspartate receptors. Pathogenic variants in GRIN2A have been associated with a wide spectrum of neurodevelopmental disorders ranging from speech disorders and/or self-limiting epilepsy (childhood epilepsy with centrotemporal spikes) to severe and disabling phenotypes (atypical childhood epilepsy with centrotemporal spikes, epileptic encephalopathy with continuous spike-wave during sleep, Landau-Kleffner syndrome and infantile-onset epileptic encephalopathy). Here we describe a family with two affected sisters with atypical childhood epilepsy with centrotemporal spikes and their mildly affected mother carrying a novel N-terminal null variant in GRIN2A gene. These familial cases corroborate previous studies showing that loss-of-function GRIN2A variants are associated with milder phenotypes, possibly due to haploinsufficiency.

Structural Determinants in the Staphylococcus aureus-Derived Phenol-Soluble Modulin α2 Peptide Required for Neutrophil Formyl Peptide Receptor Activation.

Highly pathogenic Staphylococcus aureus strains produce phenol-soluble modulins (PSMs), which are N-formylated peptides. Nanomolar concentrations of PSMα2 are recognized by formyl peptide receptor 2 (FPR2), but unlike the prototypic FPR2 agonist WKYMVM, PSMα2 is a biased signaling agonist. The truncated N-terminal PSMα2 variant, consisting of the five N-terminal residues, is no longer recognized by FPR2, showing that the C-terminal part of PSMα2 confers FPR2 selectivity, whereas the N-terminal part may interact with the FPR1 binding site. In the current study, a combined pharmacological and genetic approach involving primary human neutrophils and engineered FPR knock-in and knockout cells was used to gain molecular insights into FPR1 and FPR2 recognition of formyl peptides as well as the receptor downstream signaling induced by these peptides. In comparison with the full-length PSMα2, we show that the peptide in which the N-terminal part of PSMα2 was replaced by fMet-Ile-Phe-Leu (an FPR1-selective peptide agonist) potently activates both FPRs for production of superoxide anions and β-arrestin recruitment. A shortened analog of PSMα2 (PSMα21-12), lacking the nine C-terminal residues, activated both FPR1 and FPR2 to produce reactive oxygen species, whereas β-arrestin recruitment was only mediated through FPR1. However, a single amino acid replacement (Gly-2 to Ile-2) in PSMα21-12 was sufficient to alter FPR2 signaling to include β-arrestin recruitment, highlighting a key role of Gly-2 in conferring FPR2-biased signaling. In conclusion, we provide structural insights into FPR1 and FPR2 recognition as well as the signaling induced by interaction with formyl peptides derived from PSMα2, originating from S. aureus bacteria.

Diverse roles and modulations of IA in spinal cord pain circuits.

This review highlights recent findings of different amplitude ranges, roles, and modulations of A-type K+ currents (IA) in excitatory (GAD67-GFP-) and inhibitory (GAD67-GFP+) interneurons in mouse spinal cord pain pathways. Endogenous neuropeptides, such as TAFA4, oxytocin, and dynorphin in particular, have been reported to modulate IA in these pain pathways, but only TAFA4 has been shown to fully reverse the opposing modulations that occur selectively in LIIo GAD67-GFP- and LIIi GAD67-GFP+ interneurons following bothneuropathic and inflammatory pain. If, as hypothesized here, Kv4 subunits underlie IA in both GAD67-GFP- and GAD67-GFP+ interneurons, then IA diversity in spinal cord pain pathways may depend on the interneuron-subtype-selective expression of Kv4 auxiliary subunits with functionally different N-terminal variants. Thus, IA emerges as a good candidate for explaining the mechanisms underlying injury-induced mechanical hypersensitivity.

The Amino Terminal Domain and Modulation of Connexin36 Gap Junction Channels by Intracellular Magnesium Ions.

Electrical synapses between neurons in the mammalian CNS are predominantly formed of the connexin36 (Cx36) gap junction (GJ) channel protein. Unique among GJs formed of a number of other members of the Cx gene family, Cx36 GJs possess a high sensitivity to intracellular Mg2+ that can robustly act to modulate the strength of electrical synaptic transmission. Although a putative Mg2+ binding site was previously identified to reside in the aqueous pore in the first extracellular (E1) loop domain, the involvement of the N-terminal (NT) domain in the atypical response of Cx36 GJs to pH was shown to depend on intracellular levels of Mg2+. In this study, we examined the impact of amino acid substitutions in the NT domain on Mg2+ modulation of Cx36 GJs, focusing on positions predicted to line the pore funnel, which constitutes the cytoplasmic entrance of the channel pore. We find that charge substitutions at the 8th, 13th, and 18th positions had pronounced effects on Mg2+ sensitivity, particularly at position 13 at which an A13K substitution completely abolished sensitivity to Mg2+. To assess potential mechanisms of Mg2+ action, we constructed and tested a series of mathematical models that took into account gating of the component hemichannels in a Cx36 GJ channel as well as Mg2+ binding to each hemichannel in open and/or closed states. Simultaneous model fitting of measurements of junctional conductance, gj, and transjunctional Mg2+ fluxes using a fluorescent Mg2+ indicator suggested that the most viable mechanism for Cx36 regulation by Mg2+ entails the binding of Mg2+ to and subsequent stabilization of the closed state in each hemichannel. Reduced permeability to Mg2+ was also evident, particularly for the A13K substitution, but homology modeling of all charge-substituted NT variants showed only a moderate correlation between a reduction in the negative electrostatic potential and a reduction in the permeability to Mg2+ ions. Given the reported role of the E1 domain in Mg2+ binding together with the impact of NT substitutions on gating and the apparent state-dependence of Mg2+ binding, this study suggests that the NT domain can be an integral part of Mg2+ modulation of Cx36 GJs likely through the coupling of conformational changes between NT and E1 domains.

Structure-activity relationships of N-terminal variants of peptidomimetic tissue transglutaminase inhibitors

Tissue transglutaminase (TG2) is a multifunctional protein that catalyses protein crosslinking in the extracellular matrix, and functions as an intracellular G-protein. While both activities have been associated with human diseases, its role as a G-protein has been linked to cancer stem cell survival and maintenance of a metastatic phenotype. Recently we have shown that targeted covalent inhibitors (TCIs) can react selectively with the enzyme active site of TG2, to allosterically abolish its ability to bind GTP. In the present work, we focused on the variation of the N-terminal group of these peptidomimetic inhibitors, in order to enhance efficiency, while reducing log P and the number of rotatable bonds. This approach led to the synthesis and evaluation of 41 novel inhibitors, some of which had greatly improved efficiency and affinity for TG2 (e.g. TCI 72: KI = 1.0 μM, kinact/KI = 4.4 × 105 M−1 min−1). Molecular modelling provided a hypothetical binding mode for these TCIs. The most efficient inhibitors were evaluated further and shown to have excellent isozyme selectivity, to block GTP binding, and to have improved pharmacokinetic properties, as expected. Their biological activity was also confirmed, in a cellular invasion assay, although with less potency than expected.

Hearing Features and Cochlear Implantation Outcomes in Patients With Pathogenic MYO15A Variants: a Multicenter Observational Study.

Recessive variants in the MYO15A gene constitute an important cause of sensorineural hearing impairment (SNHI). However, the clinical features of MYO15A-related SNHI have not been systemically investigated. This study aimed to delineate the hearing features and outcomes in patients with pathogenic MYO15A variants. This study recruited 40 patients with biallelic MYO15A variants from 31 unrelated families. The patients were grouped based on the presence of N-terminal domain variants (N variants). The longitudinal audiological data and for those undergoing cochlear implantation, the auditory and speech performance with cochlear implants, were ascertained and compared between patients with different genotypes. At the first audiometric examination, 32 patients (80.0%) presented with severe to profound SNHI. Patients with at least one allele of the N variant exhibited significantly better hearing levels than those with biallelic non-N variants (78.2 ± 23.9 dBHL and 94.7 ± 22.8 dBHL, respectively) (p = 0.033). Progressive SNHI was observed in 82.4% of patients with non-profound SNHI, in whom the average progression rate of hearing loss was 6.3 ± 4.8 dBHL/year irrespective of the genotypes. Most of the 25 patients who underwent cochlear implantation exhibited favorable auditory and speech performances post-implantation. The hearing features of patients with biallelic pathogenic MYO15A variants are characterized by severe to profound SNHI, rapid hearing progression, and favorable outcomes with cochlear implants. Periodic auditory monitoring is warranted for these patients to enable early intervention.

FANCA Variants in Exons 27-30 Are Associated with Solid Tumors in Fanconi Anemia

Fanconi anemia (FA) is predominantly an autosomal recessive inherited bone marrow failure syndrome (IBMFS) characterized by congenital anomalies, bone marrow failure (BMF) and an increased cancer risk. It is caused by pathogenic variants in more than 22 genes in the FA/BRCA DNA repair pathway. Approximately 60% of patients have biallelic variants in FANCA. We sought to identify phenotypic differences and clinical outcomes of patients with diverse FANCA variants.We analyzed data from 86 patients with variants in FANCA enrolled in the National Cancer Institute (NCI) IBMFS study (ClinicalTrials.gov identifier 00027274). FANCA variants were determined through review of genetic test reports or whole exome sequencing done as part of the study. The variants were plotted using the online tool ProteinPaint (https://proteinpaint.stjude.org, Figure 1A). Clinical data were extracted from review of medical records and/or evaluations at the NIH.We compared patients with: 1) hypomorphic (hypomorphic variant on one or both alleles) versus null genotype (null variants on both alleles), 2) single nucleotide variant (SNV)/small insertions/deletions (indels) on both alleles versus SNV/small indels on one allele plus large multi-exon deletion on the other allele versus large multi-exon deletions on both alleles. We further compared patients with one or biallelic variants involving the BRCA1 interaction region in the N-terminal domain, FAAP20-binding domain, and variants in exons 27 to 30 where we saw an accumulation of variants, with patients who did not have variants in these regions. We evaluated physical abnormalities that are part of VACTERL-H (Vertebral, Anal, Cardiac, Tracheo-esophageal fistula, Esophageal or duodenal atresia, Renal, upper Limb abnormalities, Hydrocephalus) association and PHENOS (skin Pigmentation abnormalities, small Head, small Eyes, central Nervous system, Otologic abnormalities, Short stature). We focused on the presence, severity and age at BMF, and development of cancers and age at first solid tumor. Frequencies were compared by Fisher's exact test, and a multiple Cox regression model was used to estimate hazard ratio of solid tumors in patients with variants in different regions of FANCA, adjusting for age and hematopoietic cell transplant status of patients with cancer. Analyses were performed using Excel and RStudio, p-value <0.05 was considered significant.The phenotypes, BMF and cancer outcomes were similar between patients with hypomorphic and null genotypes. Similarly, comparison between patients with SNV/small indels, SNV/small indel plus large deletion, and biallelic large deletions did not reveal significant associations. Comparison according the location of the variants on FANCA protein showed that VACTERL-H and VACTERL-H plus PHENOS were less common in patients with at least one SNV/small indel in the BRCA1 interaction region of FANCA compared with patients without variants in this region (2/33 vs 12/51, p= 0.04; 1/33 vs 11/51, p= 0.024, respectively). These associations were not observed when we included patients with large deletions encompassing the BRCA1 interaction region.Eighteen of the 86 patients developed solid tumors; 15/45 patients with an SNV/small indel and/or large deletion within or including exons 27-30 region developed solid tumors compared with 3/41 patients without variants in this region (p= 0.003). Cox regression analysis showed that patients with variants within or involving exons 27-30 were at higher risk of developing solid tumors compared with those without variants in this region (HR: 6.2, 95% CI: 1.36-28.2, Figure 1B). There was no difference between the age at first cancer or type of solid tumors in patients with and without the variant involving this region. The frequency, severity, and age of BMF were also similar between the groups.Our data highlight the possibility that variants involving exons 27-30 within the C-terminal domain of FANCA may be associated with solid tumor development. FANCA forms a homodimer through the interaction between C-terminal domains; variants in this region may affect dimerization and further protein function. Functional analysis and in vivo studies of individual variants in this region and effects of the variants in trans might provide new insights into oncogenesis in FA and may have implications in personalized cancer screening. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Tryptophan (W) at position 37 of murine IL-12/IL-23 p40 is mandatory for binding to IL-12Rβ1 and subsequent signal transduction

Interleukin (IL)-12 and IL-23 are composite cytokines consisting of p35/p40 and p19/p40, respectively, which signal via the common IL-12 receptor β1 (IL-12Rβ1) and the cytokine-specific receptors IL-12Rβ2 and IL-23R. Previous data showed that the p40 component interacts with IL-12Rβ1, whereas p19 and p35 subunits solely bind to IL-23R and IL-12Rβ2, resulting in tetrameric signaling complexes. In the absence of p19 and p35, p40 forms homodimers and may induce signaling via IL-12Rβ1 homodimers. The critical amino acids of p19 and p35 required for binding to IL-23R and IL-12Rβ2 are known, and two regions of p40 critical for binding to IL-12Rβ1 have recently been identified. In order to characterize the involvement of the N-terminal region of p40 in binding to IL-12Rβ1, we generated deletion variants of the p40-p19 fusion cytokine. We found that an N-terminal deletion variant missing amino acids M23 to P39 failed to induce IL-23-dependent signaling and did not bind to IL-12Rβ1, whereas binding to IL-23R was maintained. Amino acid replacements showed that p40W37K largely abolished IL-23-induced signal transduction and binding to IL-12Rβ1, but not binding to IL-23R. Combining p40W37K with D36K and T38K mutations eliminated the biological activity of IL-23. Finally, homodimeric p40D36K/W37K/T38K did not interact with IL-12Rβ1, indicating binding of homodimeric p40 to IL-12Rβ1 is comparable to the interaction of IL-23/IL-12 and IL-12Rβ1. In summary, we have defined D36, W37, and T38 as hotspot amino acids for the interaction of IL-12/IL-23 p40 with IL-12Rβ1. Structural insights into cytokine–cytokine receptor binding are important to develop novel therapeutic strategies.

Genetic and clinical analysis of X-linked hypophosphatemic rickets

Objective: To investigate the clinical and genetic features, and treatment of X-linked hypophosphatemic rickets (XLH). Methods: In this retrospective study, we reviewed the medical records of 25 pediatric patients with XLH who were admitted to Department of Endocrinology Genetics and Metabolism,Beijing Children's Hospital from January 2010 to January 2020. The clinical characteristics, PHEX gene variants, as well as clinical outcome of the patients were summarized. To analyze the correlation between genotype and phenotype, the patients were divided into different subgroups according to the location of the variants, including N-terminal-located vs. C-terminal-located variant, and Zn-binding domain exon 17 or 19 variant vs. non-exon 17 or 19 variant. The age at onset, height standard deviation score (HtSDS), intercondylar or intermalleolar distance, fasting serum phosphorus, and HtSDS and intercondylar or intermalleolar distance at the final follow-up were compared by rank sum test or t text. Results: Among the 25 children with XLH, 8 were boys and 17 were girls. The median age of onset was 1.2 (1.0, 1.8) years, and the median age of diagnosis was 2.5 (1.5, 4.3) years. The main clinical manifestations were abnormal gait and lower limb deformity. The HtSDS was -2.0(-3.2, -0.8), and the intercondylar or intermalleolar distance was 4.5 (3.0, 6.0) cm. The fasting serum phosphorus level was 0.8 (0.7, 0.9) mmol/L, while the serum alkaline phosphatase level was (721±41) U/L and the serum calcium level was (2.5±0.1) mmol/L. Three patients (12%) had parathyroid hormone levels above the upper limit of the normal range. Twenty-five patients (100%) showed radiographic changes of active rickets. Nephrocalcinosis was found in 2 cases (9%). Twenty-four different PHEX variations were detected in 25 patients, among whom 11 (44%) had not been reported previously. No hot spot variation was found. No statistical differences (all P>0.05) were identified in clinical features and outcomes either in comparing patients with N-terminal (21 cases) and C-terminal (4 cases) variants, or in comparing patients with variant located in exon 17 or 19 (4 cases) or not (21 cases). Twenty-four cases (96%) were treated regularly with phosphate supplements and active vitamin D. After 2.7 (1.6, 5.0) years of follow-up, clinical symptoms were relieved in 96% (24/25) of the patients. The HtSDS after treatment had no significant difference compared to that before treatment (-2.0(-3.2, -0.8) vs.-2.0(-2.8, -1.1),Z =-0.156, P>0.05), while the intercondylar or intermalleolar distance after treatment was significantly reduced compared to that before treatment (4.5(3.0, 6.0) vs. 1.5(0, 3.3) cm, Z =-3.043, P<0.05). Bone X-rays were reexamined in 17 cases after treatment, and radiographic signs of rickets were improved. Eighteen cases had secondary hyperparathyroidism and 7 cases had nephrocalcinosis. Conclusions: The main clinical manifestations of XLH are abnormal gait, lower limb deformity and short stature. A high proportion of novel variations of PHEX gene but no hot spot variation neither genotype-phenotype correlation are found. Regular treatment with phosphate supplements and active vitamin D can significantly improve the symptoms except for the height. However, the rate of adverse events including secondary hyperparathyroidism and nephrocalcinosis seems to be high.

Functional analysis of novel genetic variants of NKX2-5 associated with nonsyndromic congenital heart disease.

NKX2-5, a master cardiac regulatory transcription factor was the first known genetic cause of congenital heart diseases (CHDs). To further investigate its role in CHD pathogenesis, we performed mutational screening of 285 CHD probands and 200 healthy controls. Five coding sequence variants were identified in six CHD cases (2.1%), including three in the N-terminal region (p.A61G, p.R95L, and p.E131K) and one each in homeodomain (HD) (p.A148E) and tyrosine-rich domain (p.P247A). Variant-p.A148E showed tertiary structure changes and differential DNA binding affinity of mutant compared to wild type. Two N-terminal variants-p.A61G and p.E131K along with HD variant p.A148E demonstrated significantly reduced transcriptional activity of Nppa and Actc1 promoters in dual luciferase promoter assay supported by their reduced expression in qRT-PCR. Nonetheless, variant p.R95L affected the synergy of NKX2-5 with serum response factor and TBX5 leading to significantly decreased Actc1 promoter activity depicting a distinctive role of this region. The aberrant expression of other target genes-Irx4, Mef2c, Bmp10, Myh6, Myh7, and Myocd is also observed in response to NKX2-5 variants, possibly due to the defective gene regulatory network. Severely impaired downstream promoter activities and abnormal expression of target genes due to N-terminal variants supports the emerging role of this region during cardiac-developmental pathways.

BAX mitochondrial integration is regulated allosterically by its α1-α2 loop.

The conformational changes converting BAX from an inert cytosolic monomer into the homo-oligomers that permeabilize the mitochondrial outer membrane (MOM) are crucial steps toward apoptosis. Here, we have explored the potential role of the BAX α1-α2 loop in this process by three mutagenic approaches: replacing loop segments with cognate loop regions from closely related proteins, alanine scanning and analysis of BAX α1-α2 loop missense mutations observed in tumours. Responsiveness to a death signal, such as tBID, was reduced by mutations in the N-terminal but not C-terminal half of the loop. N-terminal loop variants, which were enriched in tumours, impaired MOM integration by allosterically reducing exposure of the BAX α9 transmembrane anchor. Most C-terminal loop variants reduced BAX stability, leading to increased BAX apoptotic function in some variants. Thus, our systematic mutagenesis suggests that the two halves of the α1-α2 loop have distinct functions. We show that the N-terminal half of the loop (its first nine residues) comprises an important allosteric regulator of BAX activation by setting the proportion of MOM-integrated BAX following a death signal. The enrichment of N-terminal loop mutations in tumours indicates that they may promote tumour cell survival and underscore the loop as a target for therapeutic manipulation of BAX function.

Heterogeneity of MYO15A variants significantly determine the feasibility of acoustic stimulation with hearing aid and cochlear implant

Autosomal recessive nonsyndromic hearing loss 3 (DFNB3) mainly leads to congenital and severe-to-profound hearing impairment, which is caused by variants in MYO15A. However, audiological heterogeneity in patients with DFNB3 hinders precision medicine in hearing rehabilitation. Here, we aimed to elucidate the heterogeneity of the auditory phenotypes of MYO15A variants according to the affected domain and the feasibilities for acoustic stimulation. We conducted whole-exome sequencing for 10 unrelated individuals from seven multiplex families with DFNB3; 11 MYO15A variants, including the novel frameshift c.900delT (p.Pro301Argfs*143) and nonsense c.4879G > T (p.Glu1627*) variants, were identified. In seven probands, residual hearing at low frequencies was significantly higher in the groups with one or two N-terminal frameshift variants in trans conformation compared to that in the group without these variants. This is consistent with the 56 individuals from the previously published reports that carried a varying number of N-terminal truncating variants in MYO15A. In addition, patients with missense variants in the second FERM domain had better hearing at low frequencies than patients without these variants. Subsequently, acoustic stimulation provided by devices such as hearing aids or cochlear implants was feasible in patients with one or two N-terminal truncating variants or a second FERM missense variant. In conclusion, N-terminal or second FERM variants in MYO15A allow the practical use of acoustic stimulation through hearing aids or electroacoustic stimulation for aural rehabilitation.

PLCδ1 Plays Central Roles in the Osmotic Activation of ΔN-TRPV1 Channels in Mouse Supraoptic Neurons and in Murine Osmoregulation.

The magnocellular neurosecretory cells (MNCs) of the hypothalamus play a vital role in osmoregulation, but the mechanisms underlying MNC osmosensitivity are not fully understood. We showed previously that high osmolality activates phospholipase C (PLC) in rat MNCs in a Ca2+-dependent manner and that PLC activation is necessary for full osmotic activation of an N-terminal variant of the TRPV1 (ΔN-TRPV1) channel. We therefore hypothesized that the Ca2+-dependent δ1 isoform of PLC contributes to ΔN-TRPV1 activation and tested whether MNC function is defective in a transgenic PLCδ1 KO mouse. Water deprivation for 24 h caused greater increases in serum osmolality and losses in body weight in PLCδ1 KO mice than it did in control mice. Action potentials and ΔN-TRPV1 currents were measured in acutely isolated mouse MNCs using whole-cell patch clamp before and after exposure to hypertonic solutions. This treatment elicited a significant activation of ΔN-TRPV1 currents and an increase in firing rate in MNCs isolated from control mice, but not from PLCδ1 KO mice. Submembranous filamentous actin was measured in isolated MNCs before and after treatment with angiotensin II and hypertonic solution. Both treatments caused an increase in filamentous actin fluorescence in MNCs isolated from control mice, but both responses were significantly attenuated in MNCs from PLCδ1 KO mice. Our data demonstrate that the PLCδ1 isoform plays a key role in the activation of ΔN-TRPV1 channels and in osmosensory transduction in MNCs. This study advances our understanding of the molecular mechanisms underlying mammalian osmoregulation.SIGNIFICANCE STATEMENT Magnocellular neurosecretory cells (MNCs) of the hypothalamus play a central role in osmoregulation. We have identified a key role for the PLCδ1 isoform in the activation of ΔN-TRPV1 channels and osmosensory transduction in MNCs. The data indicate that the PLCδ1 isoform is activated by the Ca2+ influx occurring during MNC action potentials and exerts a positive feedback on ΔN-TRPV1 channels to enhance MNC excitability. This study provides evidence that PLCδ1 is a key molecule underlying osmosensory transduction, the regulation of VP release, and osmoregulation.

Functional Characterization of Rare Genetic Variants in the N-Terminus of Complement Factor H in aHUS, C3G, and AMD.

Membranoproliferative glomerulonephritis (MPGN), C3 glomerulopathy (C3G), atypical haemolytic uraemic syndrome (aHUS) and age-related macular degeneration (AMD) have all been strongly linked with dysfunction of the alternative pathway (AP) of complement. A significant proportion of individuals with MPGN, C3G, aHUS and AMD carry rare genetic variants in the CFH gene that cause functional or quantitative deficiencies in the factor H (FH) protein, an important regulator of the AP. In silico analysis of the deleteriousness of rare genetic variants in CFH is not reliable and careful biochemical assessment remains the gold standard. Six N-terminal variants of uncertain significance in CFH were identified in patients with these diseases of the AP and selected for analysis. The variants were produced in Pichia Pastoris in the setting of FH CCPs 1–4, purified by nickel affinity chromatography and size exclusion and characterized by surface plasmon resonance and haemolytic assays as well as by cofactor assays in the fluid phase. A single variant, Q81P demonstrated a profound loss of binding to C3b with consequent loss of cofactor and decay accelerating activity. A further 2 variants, G69E and D130N, demonstrated only subtle defects which could conceivably over time lead to disease progression of more chronic AP diseases such as C3G and AMD. In the variants S159N, A161S, and M162V any functional defect was below the capacity of the experimental assays to reliably detect. This study further underlines the importance of careful biochemical assessment when assigning functional consequences to rare genetic variants that may alter clinical decisions for patients.