Unraveling the molecular interactions between α7 nicotinic receptor and a RIC3 variant associated with backward speech.

Recent work putatively linked a rare genetic variant of the chaperone Resistant to Inhibitors of acetylcholinesterase (RIC3) (NM_024557.4:c.262G > A, NP_078833.3:p.G88R) to a unique ability to speak backwards, a language skill that is associated with exceptional working memory capacity. RIC3 is important for the folding, maturation, and functional expression of α7 nicotinic acetylcholine receptors (nAChR). We compared and contrasted the effects of RIC3G88R on assembly, cell surface expression, and function of human α7 receptors using fluorescent protein tagged α7 nAChR and Förster resonance energy transfer (FRET) microscopy imaging in combination with functional assays and 125I-α-bungarotoxin binding. As expected, the wild-type RIC3 protein was found to increase both cell surface and functional expression of α7 receptors. In contrast, the variant form of RIC3 decreased both. FRET analysis showed that RICG88R increased the interactions between RIC3 and α7 protein in the endoplasmic reticulum. These results provide interesting and novel data to show that a RIC3 variant alters the interaction of RIC3 and α7, which translates to decreased cell surface and functional expression of α7 nAChR.

IgG1-3 MuSK Antibodies Inhibit AChR Cluster Formation, Restored by SHP2 Inhibitor, Despite Normal MuSK, DOK7, or AChR Subunit Phosphorylation.

BACKGROUND AND OBJECTIVES: Up to 50% of patients with myasthenia gravis (MG) without acetylcholine receptor antibodies (AChR-Abs) have antibodies to muscle-specific kinase (MuSK). Most MuSK antibodies (MuSK-Abs) are IgG4 and inhibit agrin-induced MuSK phosphorylation, leading to impaired clustering of AChRs at the developing or mature neuromuscular junction. However, IgG1-3 MuSK-Abs also exist in MuSK-MG patients, and their potential mechanisms have not been explored fully. METHODS: C2C12 myotubes were exposed to MuSK-MG plasma IgG1-3 or IgG4, with or without purified agrin. MuSK, Downstream of Kinase 7 (DOK7), and ßAChR were immunoprecipitated and their phosphorylation levels identified by immunoblotting. Agrin and agrin-independent AChR clusters were measured by immunofluorescence and AChR numbers by binding of 125I-α-bungarotoxin. Transcriptomic analysis was performed on treated myotubes. RESULTS: IgG1-3 MuSK-Abs impaired AChR clustering without inhibiting agrin-induced MuSK phosphorylation. Moreover, the well-established pathway initiated by MuSK through DOK7, resulting in ßAChR phosphorylation, was not impaired by MuSK-IgG1-3 and was agrin-independent. Nevertheless, the AChR clusters did not form, and both the number of AChR microclusters that precede full cluster formation and the myotube surface AChRs were reduced. Transcriptomic analysis did not throw light on the pathways involved. However, the SHP2 inhibitor, NSC-87877, increased the number of microclusters and led to fully formed AChR clusters. DISCUSSION: MuSK-IgG1-3 is pathogenic but seems to act through a noncanonical pathway. Further studies should throw light on the mechanisms involved at the neuromuscular junction.

Motor end-plate analysis to diagnose immune-mediated myasthenia gravis in seronegative patients.

This study aimed to evaluate the diagnostic usefulness of motor end-plate (MEP) analysis along with clustered acetylcholine receptor (AChR) antibody (Ab) assays in patients with myasthenia-like symptoms but negative routine AChR and muscle-specific kinase (MuSK) Ab tests. MEP analysis of muscle biopsies of the biceps brachii was performed in 20 patients to try to differentiate between those with or without immune-mediated myasthenia gravis (MG). Using a quantitative method, complement C3 deposition and AChR densities in MEPs were examined. Independently, cell-based assays were used to detect serum clustered-AChR Abs. Only five of 20 patients had complement deposition at MEPs; four of these patients had reduced AChR densities similar to those in patients with typical AChR Ab positive MG, and distinct from those in the remaining 15 patients. Two of the four serum samples from these patients had clustered-AChR Abs. All complement-positive patients were considered as having immune-mediated MG and improved with appropriate treatments; although one patient presented with MG 3 years later, the remaining patients had other diagnoses during over 10 years of follow-up. These results suggest the usefulness of MEP analysis of muscle biopsies in diagnosing immune-mediated MG in seronegative patients with myasthenia-like symptoms but, due to the invasiveness of the muscle biopsy procedure, clustered AChR Abs should, if possible, be tested first.

Pretreatment stabilization increases completion of trauma-focused evidence-based psychotherapies.

OBJECTIVE: Veterans with posttraumatic stress disorder (PTSD) initiate and complete cognitive processing therapy (CPT) and prolonged exposure (PE) at low rates within Veterans Health Administration (VHA) despite substantial dissemination and training. This study investigated how trauma-informed, skills-based treatment ("stabilization") administered before CPT and PE was related to initiation and completion of trauma-focused evidence-based psychotherapies (TF-EBPs). METHOD: Data were extracted from the VHA electronic medical record to identify veterans who initiated outpatient treatment in the PTSD Clinical Team (PCT) at a Veterans Affairs Health Care System. Treatment initiation was defined as three or more PCT visits with no prior PCT care for at least 18 months (N = 341). Before initiation of TF-EBP, veterans received either no stabilization or received individual and/or group stabilization. RESULTS: Twenty-eight percent of veterans without stabilization (n = 115) initiated TF-EBP, compared with 34% of veterans who completed individual-only stabilization (n = 82), and 10% of veterans who completed group-only stabilization (n = 29, p = .050). Compared with those with no stabilization, individual stabilization was associated with significantly higher TF-EBP completion (93% vs. 50%, p < .001). CPT completion was also significantly higher for veterans who received individual-only stabilization (90% vs. 43%, p = .001). Results for PE followed the same relationship, but did not reach significance (100% vs. 67%, p = .090). CONCLUSIONS: Findings suggest that individual stabilization may improve delivery of TF-EBPs in VHA settings by increasing TF-EBP completion without reducing initiation, while pretreatment with group-only stabilization may reduce initiation of TF-EBPs. Results inform how models of care can improve TF-EBP retention and completion among veterans with PTSD. (PsycInfo Database Record (c) 2022 APA, all rights reserved).

Assisted Reproductive Technology Outcomes in Women With Heart Disease.

Background: Women with infertility and heart disease (HD) are increasingly seeking assisted reproductive technology (ART). There is only one other study that examines the safety profile of ART in this population. This study aims to evaluate the cardiac, reproductive, and obstetric outcomes of ART in women with HD. Methods: We conducted a retrospective case-control study of women with underlying congenital or acquired HD who underwent ART at a single University fertility center from 1/2010-3/2019. Women undergoing in-vitro fertilization (IVF), oocyte cryopreservation (OC) or embryo banking (EB) with HD were included. Cases were matched 3:1 with age-, cycle type- and cycle start date- matched controls without HD. Outcomes included cardiovascular (CV), reproductive, and obstetric complications during or following ART. Results: Twenty women with HD were included. 15 (75%) had congenital HD, 1 (5%) had valvular disease, 1 (5%) had acquired cardiomyopathy, and 3 (15%) had arrhythmias. 90% were New York Heart Association class I. 55% of HD cases were modified WHO (mWHO) risk classification 1-2, 40% were mWHO 2-3 or 3, 5% were mWHO 4. Cases underwent 25 IVF, 5 OC, and 5 EB cycles and were compared with 79 controls who underwent 174 cycles. No CV complications or deaths occurred amongst cases following ART or pregnancy. There was no difference in risk of ART or obstetric outcomes amongst cases versus controls. Conclusion: For women with HD in this small, low -risk cohort, ART posed few risks that were similar in frequency to healthy controls.

Investigation of Global Gene Expression of Human Blastocysts Diagnosed as Mosaic using Next-generation Sequencing.

Embryos are diagnosed as mosaic if their chromosomal copy number falls between euploid and aneuploid. The purpose of this study was to investigate the impact of mosaicism on global gene expression. This study included 42 blastocysts that underwent preimplantation genetic testing for aneuploidy (PGT-A) and were donated for IRB approved research. Fourteen blastocysts were diagnosed as mosaic with Next-generation Sequencing (NGS). Three NGS diagnosed euploid embryos, and 25 aneuploid embryos (9 NGS, 14 array Comparative Genomic Hybridization, 2 Single Nucleotide Polymorphism array) were used as comparisons. RNA-sequencing was performed on all of the blastocysts. Differentially expressed genes (DEGs) were calculated using DESeq2/3.5 (R Bioconductor Package) with p < 0.05 considered significantly differentially expressed. Pathway analysis was performed on mosaic embryos using EnrichR with p < 0.05 considered significant. With euploid embryo gene expression used as a control, 12 of 14 mosaic embryos had fewer DEGs compared to aneuploid embryos involving the same chromosome. On principal component analysis (PCA), mosaic embryos mapped separately from aneuploid embryos. Pathways involving cell proliferation, differentiation, and apoptosis were the most disrupted within mosaic embryos. Mosaic embryos have decreased disruption of global gene expression compared to aneuploid embryos. This study was limited by the small sample size, lack of replicate samples for each mosaic abnormality, and use of multiple different PGT-A platforms for the diagnosis of aneuploid embryos.

A rare mutation in the COLQ gene causing congenital myasthenic syndrome with remarkable improvement to fluoxetine: A case report.

Congenital myasthenic syndromes (CMS) are genetically determined heterogenous disorders of neuromuscular transmission. We report a rare mutation of COLQ causing CMS in an Asian man that remarkably improved with fluoxetine. A 51-year-old Sri Lankan man with slowly progressive fatigable muscle weakness since eight years of age, presented with type 2 respiratory failure that required mechanical ventilation in the acute crisis and subsequent home-based non-invasive ventilation. His birth and family histories were unremarkable. On examination, he had limb girdle type of muscle weakness with fatigability and normal tendon reflexes with no ocular or bulbar involvement. DNA sequencing revealed a pathogenic homozygous mutation in COLQ gene: ENST00000383788.10:exon16:c.1228C>T:p.R410W, the first report in an Asian. Treatment with fluoxetine resulted in remarkable improvement and regain of muscle power and independence from assisted ventilation.

Slow-Channel Congenital Myasthenic Syndrome due to a Novel Mutation in the Acetylcholine Receptor Alpha Subunit in a South Asian: A Case Report.

Congenital myasthenic syndromes (CMS) result from genetic mutations that cause aberrations in structure and/or function of proteins involved in neuromuscular transmission. The slow-channel CMS (SCCMS) is an autosomal dominant postsynaptic defect caused by mutations in genes encoding alpha, beta, delta, or epsilon subunits of the acetylcholine receptor resulting in a functional defect which is an increase of the opening time of the receptor. We report a case of SCCMS due to a heterozygous mutation in the M2 domain of the AChR alpha subunit - CHRNA1:ENST00000348749.6:exon7:c.806T>G:p.Val269Gly and corresponding kinetic defect. A substitution of valine with phenylalanine in the same position has been previously described. This is the first reported case of a new CHRNA1 variant in a patient with SCCMS from South Asia. We also highlight the phenotype that would favour a genetic basis over an autoimmune one, in an adult presenting with fatigable weakness.

Congenital myasthenic syndrome due to a TOR1AIP1 mutation: a new disease pathway for impaired synaptic transmission.

Congenital myasthenic syndromes are inherited disorders characterized by fatiguable muscle weakness resulting from impaired signal transmission at the neuromuscular junction. Causative mutations have been identified in genes that can affect the synaptic function or structure. We identified a homozygous frameshift deletion c.127delC, p. Pro43fs in TOR1AIP1 in two siblings with limb-girdle weakness and impaired transmission at the neuromuscular synapse. TOR1AIP1 encodes the inner nuclear membrane protein lamin-associated protein 1. On muscle biopsy from the index case, lamin-associated protein 1 was absent from myonuclei. A mouse model with lamin-associated protein 1 conditionally knocked out in striated muscle was used to analyse the role of lamin-associated protein 1 in synaptic dysfunction. Model mice develop fatiguable muscle weakness as demonstrated by using an inverted screen hang test. Electromyography on the mice revealed a decrement on repetitive nerve stimulation. Ex vivo analysis of hemi-diaphragm preparations showed both miniature and evoked end-plate potential half-widths were prolonged which was associated with upregulation of the foetal acetylcholine receptor γ subunit. Neuromuscular junctions on extensor digitorum longus muscles were enlarged and fragmented, and the number of subsynaptic nuclei was significantly increased. Following these findings, electromyography was performed on cases of other nuclear envelopathies caused by mutations in LaminA/C or emerin, but decrement on repetitive nerve stimulation or other indications of defective neuromuscular transmission were not seen. Thus, this report highlights the first nuclear membrane protein in which defective function can lead to impaired synaptic transmission.

Effect of salbutamol on neuromuscular junction function and structure in a mouse model of DOK7 congenital myasthenia.

Congenital myasthenic syndromes (CMS) are characterized by fatigable muscle weakness resulting from impaired neuromuscular transmission. ß2-adrenergic agonists are an effective treatment for DOK7-CMS. DOK7 is a component within the AGRN-LRP4-MUSK-DOK7 signalling pathway that is key for the formation and maintenance of the synaptic structure of the neuromuscular junction (NMJ). The precise mechanism of action of ß2-adrenergic agonists at the NMJ is not fully understood. In this study, we investigated whether ß2-adrenergic agonists improve both neurotransmission and structural integrity of the NMJ in a mouse model of DOK7-CMS. Ex-vivo electrophysiological techniques and microscopy of the NMJ were used to study the effect of salbutamol, a ß2-adrenergic agonist, on synaptic structure and function. DOK7-CMS model mice displayed a severe phenotype with reduced weight gain and perinatal lethality. Salbutamol treatment improved weight gain and survival in DOK7 myasthenic mice. Model animals had fewer active NMJs, detectable by endplate recordings, compared with age-matched wild-type littermates. Salbutamol treatment increased the number of detectable NMJs during endplate recording. Correspondingly, model mice had fewer acetylcholine receptor-stained NMJs detected by fluorescent labelling, but following salbutamol treatment an increased number were detectable. The data demonstrate that salbutamol can prolong survival and increase NMJ number in a severe model of DOK7-CMS.

Access to infertility care in a low-resource setting: bridging the gap through resident and fellow education in a New York City public hospital.

PURPOSE: Improving access to care is an issue at the forefront of reproductive medicine. We sought to describe how one academic center, set in the background of a large and diverse metropolitan city, cares for patients with extremely limited access to reproductive specialists. METHODS: The NYU Reproductive Endocrinology and Infertility (REI) Fellowship program provides a "fellow-run clinic" within Manhattan's Bellevue Hospital Center, which is led by the REI fellows and supervised by the REI attendings of the NYU Langone Health system. A description of the history of the hospital as well as the logistics of the fertility clinic is provided as a logistical template for implementation. RESULTS: The fellow-run fertility clinic at Bellevue hospital is held on two half days per month seeing approximately 150 new patients per year. The fertility workup, counseling, surgery, as well as ovulation induction, and early pregnancy management are offered within the construct of the fellowship and residency at NYU. Barriers to care and ways to circumvent those barriers are discussed in detail. CONCLUSION: By utilizing the ambition and construct of the OB/GYN programs, we greatly improve care for an otherwise underserved patient population by offering an efficient and optimal infertility workup and treatment in a population that would otherwise be without care. We utilize the framework of graduate medical education to provide autonomy, experience, and mentorship to both residents and fellows in our programs in an effort to provide a solution to combating inequity in infertility care.

Myasthenia gravis AChR antibodies inhibit function of rapsyn-clustered AChRs.

OBJECTIVE: Direct inhibition of acetylcholine receptor (AChR) function by autoantibodies (Abs) is considered a rare pathogenic mechanism in myasthenia gravis (MG), but is usually studied on AChRs expressed in cell lines, rather than tightly clustered by the intracellular scaffolding protein, rapsyn, as at the intact neuromuscular junction. We hypothesised that clustered AChRs would provide a better target for investigating the functional effects of AChR-Abs. METHODS: Acetylcholine-induced currents were measured using whole-cell patch clamping and a fast perfusion system to assess fast (<2 min) functional effects of the serum samples. The sensitivity, specificity and rapidity of the system were first demonstrated by applying maternal AChR-Ab positive plasmas known to inhibit fetal AChR function in TE671 cells. Eleven previously untested AChR-Ab positive MG sera, 10 AChR-Ab negative MG sera and 5 healthy control sera were then applied to unclustered and rapsyn-clustered human adult AChRs in CN21 cells. RESULTS: The maternal AChR-Ab positive plasmas reduced fetal AChR currents, but not adult AChR currents, by >80% within 100 s. Only 2/11 AChR-Ab positive sera inhibited AChR currents in unclustered AChRs, but 6/11 AChR-Ab positive sera compared with none of the 10 AChR-Ab negative sera (p=0.0020) inhibited rapsyn-clustered AChR currents, and current inhibition by the AChR-Ab positive sera was greater when the AChRs were clustered (p=0.0385). None of the sera had detectable effects on desensitisation or recovery from desensitisation. CONCLUSION: These results show that antibodies can inhibit AChR function rapidly and demonstrate the importance of clustering in exploring pathogenic disease mechanisms of MG Abs.

Clinical error rates of next generation sequencing and array comparative genomic hybridization with single thawed euploid embryo transfer.

We investigated clinical error rates with single thawed euploid embryo transfer (STEET) diagnosed by next generation sequencing (NGS) and array comparative genomic hybridization (aCGH). A total of 1997 STEET cycles after IVF with preimplantation genetic testing for aneuploidy (PGT-A) from 2010 to 2017 were identified; 1151 STEET cycles utilized NGS, and 846 STEET cycles utilized aCGH. Any abortions, spontaneous or elective, in which products of conception (POCs) were collected were reviewed. Discrepancies between chorionic villus sampling, amniocentesis, or live birth results and PGT-A diagnosis were also included. Primary outcomes were clinical error rate per: ET, pregnancy with gestational sac, live birth, and spontaneous abortion with POCs available for analysis. Secondary outcomes included implantation rate (IR), spontaneous abortion rate (SABR), and ongoing pregnancy/live birth rate (OPR/LBR). The clinical error rates in the NGS cohort were: 0.7% per embryo, 1% per pregnancy with gestational sac, and 0.1% rate per OP/LB. The error rate per SAB with POCs was 13.3%. The IR was 69.1%, the OPR/LBR was 61.6%, and the spontaneous abortion rate was 10.2%. The clinical error rates in the aCGH cohort were: 1.3% per embryo, 2% per pregnancy with gestational sac, and 0.4% rate per OP/LB. The error rate per SAB with POCs was 23.3%. The IR was 63.8%, the OPR/LBR was 54.6%, and the SAB rate was 12.4%. Our findings demonstrate that, although NGS and aCGH are sensitive platforms for PGT-A, errors still occur. Appropriate patient counseling and routine prenatal screening are recommended for all patients undergoing IVF/PGT-A.

Congenital myasthenic syndrome due to mutations in MUSK suggests that the level of MuSK phosphorylation is crucial for governing synaptic structure.

MUSK encodes the muscle-specific receptor tyrosine kinase (MuSK), a key component of the agrin-LRP4-MuSK-DOK7 signaling pathway, which is essential for the formation and maintenance of highly specialized synapses between motor neurons and muscle fibers. We report a patient with severe early-onset congenital myasthenic syndrome and two novel missense mutations in MUSK (p.C317R and p.A617V). Functional studies show that MUSK p.C317R, located at the frizzled-like cysteine-rich domain of MuSK, disrupts an integral part of MuSK architecture resulting in ablated MuSK phosphorylation and acetylcholine receptor (AChR) cluster formation. MUSK p.A617V, located at the kinase domain of MuSK, enhances MuSK phosphorylation resulting in anomalous AChR cluster formation. The identification and evidence for pathogenicity of MUSK mutations supported the initiation of treatment with ß2-adrenergic agonists with a dramatic improvement of muscle strength in the patient. This work suggests uncharacterized mechanisms in which control of the precise level of MuSK phosphorylation is crucial in governing synaptic structure.

Muscle acetylcholine receptor conversion into chloride conductance at positive potentials by a single mutation.

Charge selectivity forms the basis of cellular excitation or inhibition by Cys-loop ligand-gated ion channels (LGICs), and is essential for physiological receptor function. There are no reports of naturally occurring mutations in LGICs associated with the conversion of charge selectivity. Here, we report on a CHRNA1 mutation (α1Leu251Arg) in a patient with congenital myasthenic syndrome associated with transformation of the muscle acetylcholine receptor (AChR) into an inhibitory channel. Performing patch-clamp experiments, the AChR was found to be converted into chloride conductance at positive potentials, whereas whole-cell currents at negative potentials, although markedly reduced, were still carried by sodium. Umbrella sampling molecular dynamics simulations revealed constriction of the channel pore radius to 2.4 Å as a result of the mutation, which required partial desolvation of the ions in order to permeate the pore. Ion desolvation was associated with an energetic penalty that was compensated for by the favorable electrostatic interaction of the positively charged arginines with chloride. These findings reveal a mechanism for the transformation of the muscle AChR into an inhibitory channel in a clinical context.

ß2-Adrenergic receptor agonists ameliorate the adverse effect of long-term pyridostigmine on neuromuscular junction structure.

Acetylcholine receptor deficiency is the most common form of the congenital myasthenic syndromes, a heterogeneous collection of genetic disorders of neuromuscular transmission characterized by fatiguable muscle weakness. Most patients with acetylcholine receptor deficiency respond well to acetylcholinesterase inhibitors; however, in some cases the efficacy of acetylcholinesterase inhibitors diminishes over time. Patients with acetylcholine receptor deficiency can also benefit from the addition of a ß2-adrenergic receptor agonist to their medication. The working mechanism of ß2-adrenergic agonists in myasthenic patients is not fully understood. Here, we report the long-term follow-up for the addition of ß2-adrenergic agonists for a cohort of patients with acetylcholine receptor deficiency on anticholinesterase medication that demonstrates a sustained quantitative improvement. Coincidently we used a disease model to mirror the treatment of acetylcholine receptor deficiency, and demonstrate improved muscle fatigue, improved neuromuscular transmission and improved synaptic structure resulting from the addition of the ß2-adrenergic agonist salbutamol to the anticholinesterase medication pyridostigmine. Following an initial improvement in muscle fatiguability, a gradual decline in the effect of pyridostigmine was observed in mice treated with pyridostigmine alone (P < 0.001). Combination therapy with pyridostigmine and salbutamol counteracted this decline (P < 0.001). Studies of compound muscle action potential decrement at high nerve stimulation frequencies (P < 0.05) and miniature end-plate potential amplitude analysis (P < 0.01) showed an improvement in mice following combination therapy, compared to pyridostigmine monotherapy. Pyridostigmine alone reduced postsynaptic areas (P < 0.001) and postsynaptic folding (P < 0.01). Combination therapy increased postsynaptic area (P < 0.001) and promoted the formation of postsynaptic junctional folds (P < 0.001), in particular in fast-twitch muscles. In conclusion, we demonstrate for the first time how the improvement seen in patients from adding salbutamol to their medication can be explained in an experimental model of acetylcholine receptor deficiency, the most common form of congenital myasthenic syndrome. Salbutamol enhances neuromuscular junction synaptic structure by counteracting the detrimental effects of long-term acetylcholinesterase inhibitors on the postsynaptic neuromuscular junction. The results have implications for both autoimmune and genetic myasthenias where anticholinesterase medication is a standard treatment.

Rapsyn facilitates recovery from desensitization in fetal and adult acetylcholine receptors expressed in a muscle cell line.

KEY POINTS: The physiological significance of the developmental switch from fetal to adult acetylcholine receptors in muscle (AChRs) and the functional impact of AChR clustering by rapsyn are not well studied. Using patch clamp experiments, we show that recovery from desensitization is faster in the adult AChR isoform. Recovery from desensitization is determined by the AChR isoform-specific cytoplasmic M3-M4 domain. The co-expression of rapsyn in muscle cells induced AChR clustering and facilitated recovery from desensitization in both fetal and adult AChRs. In fetal AChRs, facilitation of recovery kinetics by rapsyn was independent of AChR clustering. These effects could be crucial adaptations to motor neuron firing rates, which, in rodents, have been shown to increase around the time of birth when AChRs cluster at the developing neuromuscular junctions. ABSTRACT: The neuromuscular junction (NMJ) is the site of a number of autoimmune and genetic disorders, many involving the muscle-type nicotinic acetylcholine receptor (AChR), although there are aspects of normal NMJ development and function that need to be better understood. In particular, there are still questions regarding the implications of the developmental switch from fetal to adult AChRs, as well as how their functions might be modified by rapsyn that clusters the AChRs. Desensitization of human muscle AChRs was investigated using the patch clamp technique to measure whole-cell currents in muscle-type (TE671/CN21) and non-muscle (HEK293) cell lines expressing either fetal or adult AChRs. Desensitization time constants were similar with both AChR isoforms but recovery time constants were shorter in cells expressing adult compared to fetal AChRs (P < 0.0001). Chimeric experiments showed that recovery from desensitization was determined by the M3-M4 cytoplasmic loops of the γ- and ε-subunits. Expression of rapsyn in TE671/CN21 cells induced AChR aggregation and also, surprisingly, shortened recovery time constants in both fetal and adult AChRs. However, this was not dependent on clustering because rapsyn also facilitated recovery from desensitization in HEK293 cells expressing a δ-R375H AChR mutant that did not form clusters in C2C12 myotubes. Thus, rapsyn interactions with AChRs lead not only to clustering, but also to a clustering independent faster recovery from desensitization. Both effects of rapsyn could be a necessary adjustment to the motor neuron firing rates that increase around the time of birth.

Should every embryo undergo preimplantation genetic testing for aneuploidy? A review of the modern approach to in vitro fertilization.

Aneuploid conceptions constitute the majority of pregnancy failures in women of advanced maternal age. The best way to combat age-related decline in fertility is through preimplantation genetic testing for aneuploidy (PGT-A). PGT-A allows for better embryo selection, which improves implantation rates with single embryo transfer and reduces miscarriage rates. Single embryo transfers decrease multiple gestations and adverse pregnancy outcomes such as preterm or low birth weight infants. Advancements in extended embryo culture, blastocyst biopsy techniques, and 24-chromosome aneuploidy screening platforms have made PGT-A safe and accessible for all patients who undergo in vitro fertilization. Improved genomic coverage of new sequencing platforms, such as next-generation sequencing, has increased the identification and diagnosis of mosaicism and partial aneuploidies in preimplantation embryos. Mosaic embryos have decreased viability compared to euploid embryos when transferred, but some mosaic embryos result in normal live births. Whole genome amplification artifacts may contribute to a misdiagnosis of mosaicism, or some mosaic embryos may self-correct to euploid after implantation. For this reason, patients without euploid embryos should be given the option of transferring mosaic embryos after genetic counseling. Further research is needed to characterize which mosaic embryos may be viable.

Next generation sequencing for preimplantation genetic screening improves pregnancy outcomes compared with array comparative genomic hybridization in single thawed euploid embryo transfer cycles.

OBJECTIVE: To evaluate whether the use of next generation sequencing (NGS) for preimplantation genetic screening (PGS) in single thawed euploid embryo transfer (STEET) cycles improves pregnancy outcomes compared with array comparative genomic hybridization (aCGH). DESIGN: Retrospective cohort study. SETTING: Single university-based fertility center. PATIENT(S): A total of 916 STEET cycles from January 2014 to December 2016 were identified. Cases included 548 STEET cycles using NGS for PGS and controls included 368 STEET cycles using aCGH for PGS. INTERVENTION(S): Patients having a STEET after undergoing IVF and PGS with either NGS or aCGH. MAIN OUTCOME MEASURE(S): Primary outcomes were implantation rate, ongoing pregnancy/live birth rate (OP/LBR), biochemical pregnancy rate (PR), and spontaneous abortion (SAB) rate. RESULT(S): The implantation rate was significantly higher in the NGS group compared with the aCGH group (71.6% vs. 64.6%). The OP/LBR was also significantly higher in the NGS group (62% vs. 54.4%), and there were significantly more biochemical pregnancies in the aCGH group compared with the NGS group (15.1% vs. 8.7%). After adjustment for confounding variables with a multiple logistic regression analysis, OP/LBR remained significantly higher in the NGS group. The SAB rate was not significantly different in the NGS group compared with the aCGH group (12.4% vs. 12.7%). CONCLUSION(S): Preimplantation genetic screening using NGS significantly improves pregnancy outcomes versus PGS using aCGH in STEET cycles. Next-generation sequencing has the ability to identify and screen for embryos with reduced viability such as mosaic embryos and those with partial aneuploidies or triploidy. Pregnancy outcomes with NGS may be improved due to the exclusion of these abnormal embryos.

Myasthenic syndromes due to defects in COL13A1 and in the N-linked glycosylation pathway.

The congenital myasthenic syndromes (CMS) are hereditary disorders of neuromuscular transmission. The number of cases recognized, at around 1:100,000 in the United Kingdom, is increasing with improved diagnosis. The advent of next-generation sequencing has facilitated the discovery of many genes that harbor CMS-associated mutations. An emerging group of CMS, characterized by a limb-girdle pattern of muscle weakness, is caused by mutations in genes that encode proteins involved in the initial steps of the N-linked glycosylation pathway, which is surprising, since this pathway is found in all mammalian cells. However, mutations in these genes may also give rise to multisystem disorders (congenital disorders of glycosylation) or muscle disorders where the myasthenic symptoms constitute only one component within a wider phenotypic spectrum. We also report a CMS due to mutations in COL13A1, which encodes an extracellular matrix protein that is concentrated at the neuromuscular junction and highlights a role for these extracellular matrix proteins in maintaining synaptic stability that is independent of the AGRN/MuSK clustering pathway. Knowledge about the neuromuscular synapse and the different proteins involved in maintaining its structure as well as function enables us to tailor treatments to the underlying pathogenic mechanisms.