R.H.M. te Morsche

Nav1.7-related small fiber neuropathy: Impaired slow-inactivation and DRG neuron hyperexcitability

Objectives: Although small fiber neuropathy (SFN) often occurs without apparent cause, the molecular etiology of idiopathic SFN (I-SFN) has remained enigmatic. Sodium channel Nav1.7 is preferentially expressed within dorsal root ganglion (DRG) and sympathetic ganglion neurons and their small-diameter peripheral axons. We recently reported the presence of Nav1.7 variants that produce gain-of-function changes in channel properties in 28% of patients with painful I-SFN and demonstrated impaired slow-inactivation in one of these mutations after expression within HEK293 cells. Here we show that the I739V Nav1.7 variant in a patient with biopsy-confirmed I-SFN impairs slow-inactivation within DRG neurons and increases their excitability. Methods: A patient with SFN symptoms including pain, and no identifiable underlying cause, was evaluated by skin biopsy, quantitative sensory testing, nerve conduction studies, screening of genomic DNA for variants in SCN9A, and functional analysis. Results: Voltage-clamp analysis following expression within DRG neurons revealed that the Nav1.7/I739V substitution impairs slow-inactivation, depolarizing the midpoint (V1/2) by 5.6 mV, and increasing the noninactivating component at 10 mV from 16.5% to 22.2%. Expression of I739V channels within DRG neurons rendered these cells hyperexcitable, reducing current threshold and increasing the frequency of firing evoked by graded suprathreshold stimuli. Conclusions: These observations provide support, from a patient with biopsy-confirmed SFN, for the suggestion that functional variants of Nav1.7 that impair slow-inactivation can produce DRG neuron hyperexcitability that contributes to pain in SFN. Nav1.7 channelopathy-associated SFN should be considered in the differential diagnosis of cases of SFN in which no other cause is found.

Familial gain-of-function Nav1.9 mutation in a painful channelopathy

Objective Gain-of-function mutations in Nav1.9 have been identified in three families with rare heritable pain disorders, and in patients with painful small-fibre neuropathy. Identification and functional assessment of new Nav1.9 mutations will help to elucidate the phenotypic spectrum of Nav1.9 channelopathies. Methods Patients from a large family with early-onset pain symptoms were evaluated by clinical examination and genomic screening for mutations in SCN9A and SCN11A. Electrophysiological recordings and multistate modelling analysis were implemented for functional analyses. Results A novel Nav1.9 mutation, p.Arg222His, was identified in patients with early-onset pain in distal extremities including joints and gastrointestinal disturbances, but was absent from an asymptomatic blood relative. This mutation alters channel structure by substituting the highly conserved first arginine residue in transmembrane segment 4 (domain 1), the voltage sensor, with histidine. Voltage-clamp recordings demonstrate a hyperpolarising shift and acceleration of activation of the p.Arg222His mutant channel, which make it easier to open the channel. When expressed in dorsal root ganglion neurons, mutant p.Arg222His channels increase excitability via a depolarisation of resting potential and increased evoked firing. Conclusions This study expands the spectrum of heritable pain disorders linked to gain-of-function mutations in Nav1.9, strengthening human validation of this channel as a potential therapeutic target for pain.

Common single nucleotide polymorphisms in transient receptor potential melastatin type 6 increase the risk for proton pump inhibitor-induced hypomagnesemia: a case–control study

Objective Proton pump inhibitors (PPIs) are effective drugs for the treatment of gastric acid-related disorders. Serious adverse events are rare for PPIs, but recent data suggest that PPIs cause hypomagnesemia. The aim of this study was to estimate the frequency of PPI-induced hypomagnesemia and to define the risk factors for its development. Materials and methods A total of 133 chronic users of PPIs were enrolled and patients were distinguished on the basis of their serum Mg2+ concentrations. Common single nucleotide polymorphisms (SNPs) in the candidate gene, transient receptor potential melastatin type 6 (TRPM6), were screened. Results Seventeen out of 133 patients had PPI-induced hypomagnesemia. The duration of PPI use was longer in those with hypomagnesemia (7.7 vs. 5.2 years). Two common SNPs in TRPM6 (rs3750425 and rs2274924) increased the risk for PPI-induced hypomagnesemia by 5.8-fold. Conclusion We found hypomagnesemia in 13% of PPI users. SNPs in TRPM6 drive the risk of developing hypomagnesemia during chronic PPI use.

Liver cyst gene knockout in cholangiocytes inhibits cilium formation and Wnt signaling

Mutations in the PRKCSH, SEC63 and LRP5 genes cause autosomal dominant polycystic liver disease (ADPLD). The proteins products of PRKCSH (alias GIIB) and SEC63 function in protein quality control and processing in the endoplasmic reticulum (ER), while LRP5 is implicated in Wnt/β-catenin signaling. To identify common denominators in the PLD pathogenesis, we mapped the PLD interactome by affinity proteomics, employing both HEK293T cells and H69 cholangiocytes. Identification of known complex members, such as glucosidase IIA (GIIA) for PRKCSH, and SEC61A1 and SEC61B for SEC63, confirmed the specificity of the analysis. GANAB, encoding GIIA, was very recently identified as an ADPLD gene. The presence of GIIA in the LRP5 complex pinpoints a potential functional connection with PRKCSH. Interestingly, all three PLD-associated protein complexes included filamin A (FLNA), a multifunctional protein described to play a role in ciliogenesis as well as canonical Wnt signalling. As ciliary dysfunction may also contribute to hereditary liver cyst formation, we evaluated the requirement of PRKCSH and SEC63 for ciliogenesis and Wnt signaling. By CRISPR/Cas9 induced knockdown of both ADPLD genes in HEK293T cells and H69 cholangiocytes, we identified that their depletion results in defective ciliogenesis. However, only H69 knockouts displayed reduced Wnt3a activation. Our results suggest that loss of PRKCSH and SEC63 leads to general defects in ciliogenesis, while quenching of the Wnt signaling cascade is cholangiocyte-restricted. Interactions of all three PLD-associated protein complexes with FLNA may mark a common link between the ADPLD proteins and the cystogenic processes driving this disease.

Hepatic cyst penetration of cefazolin in patients receiving aspiration sclerotherapy

BACKGROUND Hepatic cyst infection is a potentially severe complication in cystic disease. Treatment demands effective antibiotic concentrations within the infected cyst. OBJECTIVES The aim of this study was to use elective hepatic cyst drainage as a unique pharmacokinetic model to investigate whether cefazolin, a first-generation cephalosporin, is able to penetrate hepatic cysts. PATIENTS AND METHODS Patients scheduled to undergo percutaneous aspiration sclerotherapy of a symptomatic non-infected, non-neoplastic hepatic cyst were eligible for this study. All participants received a single perioperative prophylactic dose of cefazolin (1000 mg, intravenously). We collected blood and cyst fluid samples to determine total and unbound cefazolin concentrations using HPLC. The primary outcome was hepatic cyst penetration, expressed as the ratio (%) of unbound concentration of cefazolin in cyst fluid to plasma (both in mg/L). RESULTS We included eight patients [male = 25%, median age = 60 years (IQR 54-75), median estimated glomerular filtration rate = 97 mL/min/1.73 m(2) (IQR 67-102) and median serum albumin = 40 g/L (IQR 37-40)]. We detected low concentrations of unbound cefazolin in cyst fluid (≤1.0 mg/L). The median plasma unbound cefazolin peak level (immediately after cefazolin administration) was 36.6 mg/L (IQR 23.7-54.1) and the level at the time of cyst fluid aspiration was 16.1 mg/L (IQR 13.0-20.1). In total, the hepatic cyst penetration of free cefazolin was only 2.2% (IQR 0.7-5.2). CONCLUSIONS We developed a study model to investigate the penetration of antibiotics into hepatic cysts. Cefazolin did not reach adequate intracystic concentrations. Future studies should explore alternatives.

Inulin significantly improves serum magnesium levels in proton pump inhibitor‐induced hypomagnesaemia

Proton pump inhibitors (PPI) are among the most widely prescribed drugs to treat gastric acid‐related disorders. PPI‐induced hypomagnesaemia, a defect in intestinal absorption of Mg2+, can be a severe side effect of chronic PPI use.