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Gestational trophoblastic diseases derived from the chorionic-type intermediate trophoblast include benign placental site nodule (PSN) and malignant epithelioid trophoblastic tumor (ETT). Among PSN, the WHO classification introduced a new entity named atypical placental site nodule (APSN), corresponding to an ETT precursor, for which the diagnostic criteria remain unclear, leading to a risk of over-diagnosis and difficulties in patient management. We retrospectively studied 8 PSN, 7 APSN and 8 ETT to better characterize this new entity. We performed an immunohistochemical analysis (p63, hPL, Cyclin E, and Ki67), a transcriptional analysis using the Nanostring method to quantify the expression of 760 genes involved in the main tumorigenesis pathways, and a RNA sequencing to identify fusion transcripts. The immunohistochemical analysis did not reveal any significant difference in Cyclin E expression between the three groups (p = 0.476), whereas the Ki67 index was significantly (p LPCAT1-TERT fusion transcripts previously reported in ETT. The transcriptomic analysis allowed robust clustering of ETT distinct from the APSN/PSN group but failed to distinguish APSN from PSN. Indeed, only seven genes were differentially-expressed between PSN and APSN samples, CCL19 upregulation and EPCAM downregulation were the most discriminating features of APSN. In contrast, 80 genes discriminated ETT from APSN, establishing a molecular signature for ETT. Gene set analysis identified significant enrichments in the DNA damage repair, immortality and stemness, and cell cycle signaling pathways when comparing ETT and APSN. These results suggested that APSN might not represent a distinct entity but rather a variant of PSN or a transitional stage between PSN and ETT. RNA sequencing and the transcriptional signature of ETT described herein could serve as triage for APSN from curettage or biopsy material, enabling the identification of the cases that need further clinical investigations.

Giant axonal neuropathy (GAN) is an autosomal recessive disease caused by mutations in the GAN gene encoding gigaxonin. Patients develop a progressive sensorimotor neuropathy affecting peripheral nervous system (PNS) and central nervous system (CNS). Methods: In this multicenter observational retrospective study, we recorded French patients with GAN mutations, and 10 patients were identified. Mean age of patients was 9.7 years (2-18), eight patients were female (80%), and all patients met infant developmental milestones and had a family history of consanguinity. Mean age at disease onset was 3.3 years (1-5), and progressive cerebellar ataxia and distal motor weakness were the initial symptoms in all cases. Proximal motor weakness and bulbar symptoms appeared at a mean age of 12 years (8-14), and patients used a wheelchair at a mean age of 16 years (14-18). One patient died at age 18 years from aspiration pneumonia. In all cases, nerve conduction studies showed a mixed demyelinating and axonal sensorimotor neuropathy and MRI showed brain and cerebellum white matter abnormalities. Polyneuropathy and encephalopathy both aggravated during the course of the disease. Patients also showed a variety of associated findings, including curly hair (100% of cases), pes cavus (80%), ophthalmic abnormalities (30%), and scoliosis (30%). Five new GAN mutations were found, including the first synonymous mutation and a large intragenic deletion. Our findings expand the genotypic spectrum of GAN mutations, with relevant implications for molecular analysis of this gene, and confirm that GAN is an age-related progressive neurodegenerative disease involving PNS and CNS.

Key points Missense mutations in the gene encoding the α1 subunit of the skeletal muscle voltage-gated Ca2+ channel induce type 1 hypokalaemic periodic paralysis, a poorly understood neuromuscular disease characterized by episodic attacks of paralysis associated with low serum K+. Acute expression of human wild-type and R1239H HypoPP1 mutant α1 subunits in mature mouse muscles showed that R1239H fibres displayed Ca2+ currents of reduced amplitude and larger resting leak inward current increased by external acidification. External acidification also produced intracellular acidification at a higher rate in R1239H fibres and inhibited inward rectifier K+ currents. These data suggest that the R1239H mutation induces an elevated leak H+ current at rest flowing through a gating pore and could explain why paralytic attacks preferentially occur during the recovery period following muscle exercise. Abstract Missense mutations in the gene encoding the α1 subunit of the skeletal muscle voltage-gated Ca2+ channel induce type 1 hypokalaemic periodic paralysis, a poorly understood neuromuscular disease characterized by episodic attacks of paralysis associated with low serum K+. The present study aimed at identifying the changes in muscle fibre electrical properties induced by acute expression of the R1239H hypokalaemic periodic paralysis human mutant α1 subunit of Ca2+ channels in a mature muscle environment to better understand the pathophysiological mechanisms involved in this disorder. We transferred genes encoding wild-type and R1239H mutant human Ca2+ channels into hindlimb mouse muscle by electroporation and combined voltage-clamp and intracellular pH measurements on enzymatically dissociated single muscle fibres. As compared to fibres expressing wild-type α1 subunits, R1239H mutant-expressing fibres displayed Ca2+ currents of reduced amplitude and a higher resting leak inward current that was increased by external acidification. External acidification also produced intracellular acidification at a higher rate in R1239H fibres and inhibited inward rectifier K+ currents. These data indicate that the R1239H mutation induces an elevated leak H+ current at rest flowing through a gating pore created by the mutation and that external acidification favours onset of muscle paralysis by potentiating H+ depolarizing currents and inhibiting resting inward rectifier K+ currents. Our results could thus explain why paralytic attacks preferentially occur during the recovery period following intense muscle exercise.

The V876E mutation in the muscle voltage-gated Ca2+ channel induces hypokalemic periodic paralysis. Fuster et al. show that the mutation generates strong resting inward leak Na+ currents even though it is not located in the voltage sensor segments., Type 1 hypokalemic periodic paralysis (HypoPP1) is a poorly understood genetic neuromuscular disease characterized by episodic attacks of paralysis associated with low blood K+. The vast majority of HypoPP1 mutations involve the replacement of an arginine by a neutral residue in one of the S4 segments of the α1 subunit of the skeletal muscle voltage-gated Ca2+ channel, which is thought to generate a pathogenic gating pore current. The V876E HypoPP1 mutation has the peculiarity of being located in the S3 segment of domain III, rather than an S4 segment, raising the question of whether such a mutation induces a gating pore current. Here we successfully transfer cDNAs encoding GFP-tagged human wild-type (WT) and V876E HypoPP1 mutant α1 subunits into mouse muscles by electroporation. The expression profile of these WT and V876E channels shows a regular striated pattern, indicative of their localization in the t-tubule membrane. In addition, L-type Ca2+ current properties are the same in V876E and WT fibers. However, in the presence of an external solution containing low-Cl− and lacking Na+ and K+, V876E fibers display an elevated leak current at negative voltages that is increased by external acidification to a higher extent in V876E fibers, suggesting that the leak current is carried by H+ ions. However, in the presence of Tyrode’s solution, the rate of change in intracellular pH produced by external acidification was not significantly different in V876E and WT fibers. Simultaneous measurement of intracellular Na+ and current in response to Na+ readmission in the external solution reveals a rate of Na+ influx associated with an inward current, which are both significantly larger in V876E fibers. These data suggest that the V876E mutation generates a gating pore current that carries strong resting Na+ inward currents in physiological conditions that are likely responsible for the severe HypoPP1 symptoms associated with this mutation.

La Paralysie Periodique Hypokaliemique de type 1 (HypoPPl) est une maladie neuromusculaire caracterisee par des crises de paralysies musculaires transitoires pouvant durer de quelques heures a quelques jours et associees a une hypokaliemie. Ces paralysies peuvent etre declenchees suite a un exercice intense, une consommation importante d’hydrates de carbone, une exposition au froid ou un stress [].

Charcot-Marie-Tooth disease (CMT) refers to a group of clinically and genetically heterogeneous inherited neuropathies. Ganglioside-induced differentiation-associated protein 1 GDAP1-related CMT has been reported in an autosomal dominant or recessive form in patients presenting either axonal or demyelinating neuropathy. We report two Sri Lankan sisters born to consanguineous parents and presenting with a severe axonal sensorimotor neuropathy. The early onset of the disease, the distal and proximal weakness and atrophy leading to major disability, along with areflexia, and, most notably, vocal cord and diaphragm paralysis were highly evocative of a GDAP1-related CMT. However, sequencing of the coding regions of the gene was normal. Whole-exome sequencing (WES) was performed and revealed that the largest region of homozygosity was around GDAP1 with several variants, mostly in non-coding regions. In view of the high clinical suspicion of GDAP1 gene involvement, we examined the variants in this gene and this, along with functional studies, allowed us to identify an alternative splicing site revealing a cryptic in-frame stop codon in intron 4 responsible for a severe loss of wild-type GDAP1. This work is the first to describe a deleterious mutation in GDAP1 gene outside of coding sequences or intronic junctions and emphasizes the importance of interpreting molecular analysis, and in particular WES results, in light of the clinical and electrophysiological phenotype.

Missense mutations in the gene encoding the α1 subunit of the skeletal muscle voltage-gated CaMissense mutations in the gene encoding the α1 subunit of the skeletal muscle voltage-gated Ca

Missense mutations in the gene encoding the alpha1 subunit of the L-type calcium channel CaV1.1 induce type 1 Hypokalemic Periodic Paralysis (HypoPP1). These mutations mainly occur at arginine residues in the fourth transmembrane segment of voltage-sensor domains. Very few studies have investigated the acute effects of these mutations on channel function and muscle membrane electrical properties because of the difficulty to express Cav1.1 in heterologous systems. In the present study we successfully transferred by electroporation the genes encoding the turboGFP-tagged human wildtype (WT) and R1239H HypoPP1 mutant Cav1.1 into hind limb mouse muscles. The expression profile of the two channels showed a regular striated pattern indicative of the localization of the channels in the t-tubule membrane. Measurement of the L-type current using the silicone-clamp technique showed that the maximal conductance and the voltage-dependence of the Cav1.1 channel were significantly reduced and shifted towards negative potentials respectively in fibers expressing R1239H Cav1.1 as compared to fibers expressing WT Cav1.1. Applying voltage ramps from a holding potential of 0, −20, −40 or −60 mV to −120 mV in the presence of an external low-chloride, sodium-free and potassium-free solution revealed a significant higher leak conductance measured between −80 and −120 mV in fibers expressing R1239H Cav1.1. Acidification of the external solution significantly increased the leak inward current, leak conductance and the fluorescence of an internally loaded pH indicator in fibers expressing R1239H. These data suggest that an elevated leak inward current, likely carrying protons, flows at resting membrane potentials in fibers expressing R1239H Cav1.1 and that acidification of the external medium following muscle exercise could contribute to favor the onset of muscle paralysis in HypoPP1.