Loss-of-function mutations of SCN10A encoding NaV1.8 α subunit of voltage-gated sodium channel in patients with human kidney stone disease

Human kidney stone disease (KSD) causes significant morbidity and public health burden worldwide. The etiology of KSD is heterogeneous, ranging from monogenic defects to complex interaction between genetic and environmental factors. However, the genetic defects causing KSD in the majority of affected families are still unknown. Here, we report the discovery of mutations of SCN10A, encoding NaV1.8 α subunit of voltage-gated sodium channel, in families with KSD. The region on chromosome 3 where SCN10A locates was initially identified in a large family with KSD by genome-wide linkage analysis and exome sequencing. Two mutations (p.N909K and p.K1809R) in the same allele of SCN10A co-segregated with KSD in the affected family. Additional mutation (p.V1149M) of SCN10A was identified in another affected family, strongly supporting the causal role of SCN10A for KSD. The amino acids at these three positions, N909, K1809, and V1149, are highly conserved in vertebrate evolution, indicating their structural and functional significances. NaV1.8 α subunit mRNA and protein were found to express in human kidney tissues. The mutant proteins expressed in cultured cells were unstable and causing reduced current density as analyzed by whole-cell patch-clamp technique. Thus, loss-of-function mutations of SCN10A were associated with KSD in the families studied.

. Segregation testing of KSD and TTYH2 variation. p.M177I variation of TTYH2 was genotyped by dCAPS technique in 4 affected families.            The patient had strong clinical history as justified from the presence of several symptoms associated with kidney stone, especially hematuria and stone passage. 2 The patient had positive results of kidney-ureter-bladder (KUB) radiography. * Age at the onset of kidney stone disease.  No significant difference was found between wild-type and mutant Na V 1.8 channels.    Genetic variations acquired from exome sequencing data in the chromosomal regions with high LOD scores (>2.80) were taken for the analysis. The variations outside exonic regions were initially excluded as they were likely to be non-disease causing polymorphisms. Since KSD in the UBRS082 family was inherited as autosomal dominant mode, the genetic variations that were shared in the two affected family members but not observed in the unaffected family member were selected for further analyses. The variations in exons that caused non-synonymous changes, stop gain/loss variants or short insertions or deletions (indels) were considered for further analysis.
The possible impacts of amino acid changes on structure and function of protein were predicted by using 6 web-based programs: Polymorphism Phenotyping v2 (PolyPhen-2), 2 VarioWatch, 3 MutationTaster, 4 Sorting Intolerant From Tolerant (SIFT), 5 MutationAssessor, 6 and Likelihood Ratio Test (LRT). 7 The impact of exon-intron boundaries on mRNA splicing process was evaluated using ESEfinder 2.0. 8 Multiple amino acid sequence alignment of Na V 1.8 α subunit of voltage-gated sodium channel from human, chimpanzee, orangutans, gibbon, dog, cow, mouse, rat, anole and chicken were carried out by using ClustalW2 program.

Genotyping of genetic variations in family members and normal control subjects
Nucleotide sequences of the genes of interest were acquired from the GenBank database for designing polymerase chain reaction (PCR) primers (Table S10) Table S12. The RT-PCR products from kidney tissues were further analyzed by Sanger DNA sequencing.

Western-blot analysis
Proteins were extracted from human kidney tissue sections (20-μm thick) in 2x SDS buffer or from transfected cells in RIPA. Ten μl of human kidney tissue protein or 15 μl of protein from transfected cells of each sample was separated by SDS-polyacrylamide gel electrophoresis (PAGE). After electrophoresis, the proteins were transferred onto a nitrocellulose membrane by following a standard protocol. The membrane was blocked with 5% skim milk in TBST solution and incubated with rabbit anti-human Na V 1.

Plasmid constructs, cell culture and transfections
The wild-type constructs, pcDNA5_Na V 1.8 and pcDNA5_Na V 1.8-venus, and reported gain-offunction mutation, pcDNA5_Na V 1.8_I1706V, were previously reported. 12, 13 The wild-type constructs were used as templates to generate mutant constructs by PCR and site-directed mutagenesis method using Pfx

Immunohistochemistry staining
Human kidney tissues from remaining biopsy specimens were fixed in 4% paraformaldehyde and embedded in paraffin blocks. Then, 4-m-thick tissues were serially cut from paraffin blocks and mounted onto glass slides coated with 1% (W/V) gelatin solution. After deparaffinization and rehydration, the kidney tissue sections were placed in sodium citrate buffer (10 mM sodium citrate, 2 mM EDTA, 0.05% tween-20, pH 6.0) and subjected to heat retrieval at 70C for 20 minutes. The sections were allowed to cool to room temperature and endogenous peroxidase activity was blocked by 3% hydrogen peroxide for 30 minutes. The sections were placed in 2% BSA for 30 minutes to block nonspecific bindings and incubated with a rabbit antibody against either Na V 1.

Double immunofluorescence staining
The human fresh-frozen kidney tissues were sliced into 2-4 μm thickness by a rotary microtome.
The tissue section was placed on a glass slide and fixed with acetone for 10 minutes before incubation with rabbit anti-human Na V 1.8 α subunit of voltage-gated sodium channel (Alomone, Jerusalem, Israel) and mouse anti-alpha 1 Na + /K + ATPase (Abcam, Cambridge, UK) as the primary antibodies, followed by incubation with donkey anti-rabbit IgG conjugated with Alexa 488 fluorescein and goat anti-mouse IgG

Electrophysiological study
Transfected cells, which had been in 200 µg/ml G418 and 1 mM lidocaine for 24 hours (48 hours after transfection), were plated onto poly-L-lysine-coated cover slips (1x10 5 cells per 35-mm Petri dish; lidocaine was still present), and left overnight (10-12 hours). Then, they were exposed to culture media without lidocaine for at least three hours before subjected to an electrophysiological experiment. Whole-cell patch-clamp recording was conducted at room temperature (25-26°C), using an Axopatch 200B patch-clamp amplifier (Axon Instruments, Foster City, CA, USA) and a Digidata 1440A analog-to-digital converter (Axon Instruments). The pipette solution contained 120 mM CsF, 15 mM NaCl, 10 mM HEPES, and 10 mM EGTA (pH 7.25 with CsOH). The extracellular bath solution contained 140 mM NaCl, 4.7 mM KCl, 1 mM CaCl 2 , 1.3 mM MgCl 2 , 5 mM HEPES, and 11 mM glucose (pH 7.4 with NaOH). To block endogenous tetrodotoxin (TTX)-sensitive Na + currents, 0.3 µM TTX was always included in the bath solution to block tetrodotoxin-sensitive (Na V 1.7, Na V 1.2, Na V 1.3) and the tetrodotoxin-resistant Na V 1.5 which is expressed at low and variable levels in these cells 14 and would be 60% inactivated at -80 mV, according to Wang et al., 2015. 15 Pipette resistance was 3.50 ± 0.08 MΩ (mean ± SEM), yielding maximal voltage errors of -2.46 ± 0.10 mV. Pipette potential was adjusted to zero before seal formation; liquid junction potential was not corrected. Holding potential (V h ) was -80 mV.
Currents were measured after leak subtraction. Current-voltage (I-V) curves were constructed from peak currents in response to a series of 100-ms step depolarization, at 5 s intervals, from -80 mV V h to potentials between -60 and +60 mV, in 10 mV increments. Current density was calculated by normalizing peak currents with cell capacitance. Conductance (G) at each voltage (V) was calculated using the equation G = I / (V-V rev ) (I, peak current; V rev , reversal potential). Individual conductance-voltage curve was plotted and fitted with a Boltzmann equation: where conductance (G) is a function of the membrane potential (V); V 1/2 is the half maximal activation voltage; and k is the slope factor. Average V 1/2 and k of each group was used to construct the group's conductance-voltage curve.
Steady-state fast inactivation was assessed with a series of 500-ms prepulses (-90 to +10 mV in 5-mV increments) followed by a 40-ms test pulse to 0 mV to assess the available channels. Slow inactivation was assessed with 30-s prepulses at potentials ranging from -110 to +20 mV, followed by a 30-ms hyperpolarization at -80 mV to allow channel recovery from fast inactivation, and a 50-ms test pulse to 0 mV. Peak inward currents obtained during test pulses of steady-state fast-inactivation and slow-inactivation protocols were normalized to maximal peak current and fitted with the Boltzmann equation as a function of the inactivating voltage. Steady-state inactivation curves were constructed from average V 1/2 and k of each group.
The time constant of inactivation was obtained by fitting decaying currents with the standard exponential equation: