The neuromuscular junction (NMJ) is one of the best-studied cholinergic synapses. Inherited defects of peripheral neurotransmission result in congenital myasthenic syndromes (CMSs), a clinically and genetically heterogeneous group of rare diseases with fluctuating fatigable muscle weakness as the clinical hallmark. Whole-exome sequencing and Sanger sequencing in six unrelated families identified compound heterozygous and homozygous mutations in SLC5A7 encoding the presynaptic sodium-dependent high-affinity choline transporter 1 (CHT), which is known to be mutated in one dominant form of distal motor neuronopathy (DHMN7A). We identified 11 recessive mutations in SLC5A7 that were associated with a spectrum of severe muscle weakness ranging from a lethal antenatal form of arthrogryposis and severe hypotonia to a neonatal form of CMS with episodic apnea and a favorable prognosis when well managed at the clinical level. As expected given the critical role of CHT for multisystemic cholinergic neurotransmission, autonomic dysfunctions were reported in the antenatal form and cognitive impairment was noticed in half of the persons with the neonatal form. The missense mutations induced a near complete loss of function of CHT activity in cell models. At the human NMJ, a delay in synaptic maturation and an altered maintenance were observed in the antenatal and neonatal forms, respectively. Increased synaptic expression of butyrylcholinesterase was also observed, exposing the dysfunction of cholinergic metabolism when CHT is deficient in vivo. This work broadens the clinical spectrum of human diseases resulting from reduced CHT activity and highlights the complexity of cholinergic metabolism at the synapse.

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De novo variants (DNVs) cause many genetic diseases. When DNVs are examined in the whole coding regions of genes in next-generation sequencing analyses, pathogenic DNVs often cluster in a specific region. One such region is the last exon and the last 50 bp of the penultimate exon, where truncating DNVs cause escape from nonsense-mediated mRNA decay [NMD(À) region]. Such variants can have dominant-negative or gain-of-function effects. Here, we first developed a resource of rates of truncating DNVs in NMD(À) regions under the null model of DNVs. Utilizing this resource, we performed enrichment analysis of truncating DNVs in NMD(À) regions in 346 developmental and epileptic encephalopathy (DEE) trios. We observed statistically significant enrichment of truncating DNVs in semaphorin 6B (SEMA6B) (p value: 2.8 3 10 À8 ; exome-wide threshold: 2.5 3 10 À6 ). The initial analysis of the 346 individuals and additional screening of 1,406 and 4,293 independent individuals affected by DEE and developmental disorders collectively identified four truncating DNVs in the SEMA6B NMD(À) region in five individuals who came from unrelated families (p value: 1.9 3 10 À13 ) and consistently showed progressive myoclonic epilepsy. RNA analysis of lymphoblastoid cells established from an affected individual showed that the mutant allele escaped NMD, indicating stable production of the truncated protein. Importantly, heterozygous truncating variants in the NMD(þ) region of SEMA6B are observed in general populations, and SEMA6B is most likely loss-of-function tolerant. Zebrafish expressing truncating variants in the NMD(À) region of SEMA6B orthologs displayed defective development of brain neurons and enhanced pentylenetetrazole-induced seizure behavior. In summary, we show that truncating DNVs in the final exon of SEMA6B cause progressive myoclonic epilepsy.

Summary:Purpose: The aim of this study was to investigate the long-term outcome of children with benign partial epilepsy in infancy (BPEI).Methods: A telephone-interview survey using a structured questionnaire was conducted with patients who were diagnosed as having possible BPEI at age 2 years and who were 8 years or older at the time of the survey. The data from 39 of 48 patients were available. The median age at the time of the survey was 11.3 years; 18 boys and 21 girls were included.Results: Three patients had a recurrence of unprovoked seizure beyond age 2 years. Four patients had cognitive problems (mild mental retardation in three and Asperger syndrome in one). An association of paroxysmal kinesigenic choreoathetosis was observed in three patients, and another three had experienced seizures associated with mild gastroenteritis. Major behavioral problems were not recognized in any patients. Four patients were excluded from having definite BPEI at age 5 years, and another two were excluded for having definite BPEI at the last follow-up. Eventually, 33 of 39 patients were categorized as having definite BPEI beyond 8 years of age.Conclusions: A large majority of patients diagnosed as possibly having BPEI at age 2 years did not have a recurrence of unprovoked seizures and cognitive problems beyond 8 years of age.

Cornelia de Lange syndrome (CdLS) is a rare multisystem disorder with specific dysmorphic features. Pathogenic genetic variants encoding cohesion complex subunits and interacting proteins (e.g., NIPBL, SMC1A, SMC3, HDAC8, and RAD21) are the major cause of CdLS. However, there are many clinically diagnosed cases of CdLS without pathogenic variants in these genes. To identify further genetic causes of CdLS, we performed whole exome sequencing in 57 CdLS families, systematically evaluating both single nucleotides variants (SNVs) and copy number variations (CNVs). We identified pathogenic genetic changes in 36 out of 57 (63.2 %) families, including 32 SNVs and four CNVs. Two known CdLS genes, NIPBL and SMC1A, were mutated in 23 and two cases, respectively. Among the remaining 32 individuals, four genes (ANKRD11, EP300, KMT2A, and SETD5) each harbored a pathogenic variant in a single individual. These variants are known to be involved in CdLS-like. Furthermore, pathogenic CNVs were detected in NIPBL, MED13L, and EHMT1, along with pathogenic SNVs in ZMYND11, MED13L, and PHIP. These three latter genes were involved in diseases other than CdLS and CdLS-like. Systematic clinical evaluation of all patients using a recently proposed clinical scoring system showed that ZMYND11, MED13L, and PHIP abnormality may cause CdLS or CdLS-like.

The catalytic subunits of acetylcholinesterase (AChE) are anchored in the basal lamina of the neuromuscular junction using a collagen-like tail subunit (ColQ) encoded by COLQ. Mutations in COLQ cause endplate AChE deficiency. An A-to-G mutation predicting p.E415G in COLQ exon 16 identified in a patient with endplate AChE deficiency causes exclusive skipping of exon 16. RNA affinity purification, mass spectrometry, and siRNA-mediated gene knocking down disclosed that the mutation disrupts binding of a splicing-enhancing RNA-binding protein, SRSF1, and de novo gains binding of a splicing-suppressing RNA-binding protein, hnRNP H. MS2-mediated artificial tethering of each factor demonstrated that SRSF1 and hnRNP H antagonistically modulate splicing by binding exclusively to the target in exon 16. Further analyses with artificial mutants revealed that SRSF1 is able to bind to degenerative binding motifs, whereas hnRNP H strictly requires an uninterrupted stretch of poly(G). The mutation compromised splicing of the downstream intron. Isolation of early spliceosome complex revealed that the mutation impairs binding of U1-70K (snRNP70) to the downstream 5′ splice site. Global splicing analysis with RNA-seq revealed that exons carrying the hnRNP H-binding GGGGG motif are predisposed to be skipped compared to those carrying the SRSF1-binding GGAGG motif in both human and mouse brains.

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Molecular hydrogen ameliorates MCT-induced PAH in rats by suppressing macrophage accumulation, reducing oxidative stress and modulating the STAT3/NFAT axis.