- Primary research
- Open Access
Identification and functional characterization of two novel mutations in KCNJ10 and PI4KB in SeSAME syndrome without electrolyte imbalance
Human Genomics volume 13, Article number: 53 (2019)
Dysfunction in inwardly rectifying potassium channel Kir4.1 has been implicated in SeSAME syndrome, an autosomal-recessive (AR), rare, multi-systemic disorder. However, not all neurological, intellectual disability, and comorbid phenotypes in SeSAME syndrome can be mechanistically linked solely to Kir4.1 dysfunction.
We therefore performed whole-exome sequencing and identified additional genetic risk-elements that might exert causative effects either alone or in concert with Kir4.1 in a family diagnosed with SeSAME syndrome.
Two variant prioritization pipelines based on AR inheritance and runs of homozygosity (ROH), identified two novel homozygous variants in KCNJ10 and PI4KB and five rare homozygous variants in PVRL4, RORC, FLG2, FCRL1, NIT1 and one common homozygous variant in HSPA6 segregating in all four patients. The novel mutation in KCNJ10 resides in the cytoplasmic domain of Kir4.1, a seat of phosphatidylinositol bisphosphate (PIP2) binding. The mutation altered the subcellular localization and stability of Kir4.1 in patient-specific lymphoblastoid cells (LCLs) compared to parental controls. Barium-sensitive endogenous K+ currents in patient-specific LCLs using whole-cell patch-clamp electrophysiology revealed membrane depolarization and defects in inward K+ ion conductance across the membrane, thereby suggesting a loss-of-function effect of KCNJ10 variant.
Altogether, our findings implicate the role of new genes in SeSAME syndrome without electrolyte imbalance and thereby speculate the regulation of Kir4.1 channel activity by PIP2 and integrin-mediated adhesion signaling mechanisms.
Channelopathies are a heterogeneous group of disorders resulting in dysfunction of ion channels. They disrupt the brain function resulting in seizures and developmental delay [1,2,3,4,5,6,7,8]. The cells of the central and peripheral nervous system contain a plethora of ion channel proteins which interact with multiple signaling pathways linking channel physiology to neuronal differentiation, axonal integrity and cell migration [6, 7, 9, 10]. Nevertheless, not all phenotypes manifested in a syndromic disorder can be attributed to monogenic variants in membrane ion channels . Therefore, for a complete molecular understanding of channelopathies, it is imperative to focus on other classes of risk-associated rare variants especially in minor genes which modify the effect of major gene mutations. Such an approach for SeSAME syndrome, a rare autosomal recessive, multisystemic neuropsychiatric illness has not been addressed and will greatly benefit to understand the etiology of Kir4.1 channel dysfunction that will ultimately inform treatment.
SeSAME syndrome (OMIM#612780), characterized by seizures, sensorineural deafness, ataxia, mental retardation, and electrolyte imbalance, otherwise known as EAST (Epilepsy, Ataxia, Sensorineural deafness, Tubulopathy) syndrome is predominantly caused by homozygous or compound heterozygous mutations in KCNJ10 gene [12, 13] encoding Kir4.1, an inwardly rectifying potassium channel. Till date, 21 mutations from 27 patients have been reported, of which 11 were from consanguineous unions . Dysfunction of Kir4.1 has been associated with other neurodegenerative conditions like amyotrophic lateral sclerosis , idiopathic epilepsy , autism spectrum disorder with seizures [16, 17], Huntington’s disease , multiple sclerosis , and Rett syndrome . Several modern-day mammals like Jack Russell Terriers, Belgian Shepherd dogs , and Malinois dogs  experienced SeSAME-like phenotype with KCNJ10 mutations.
Kir 4.1 channels display greater inward K+ flow at negative resting membrane potential to equilibrium potential for K+ (Ek), while at more positive membrane potentials, outward flow of K+ is inhibited by intracellular Mg2+ and polyamines . Depending on tissue localization and assembly of Kir4.1 subunit, these channels exhibit distinctive physiological properties . Kir4.1 channel play conspicuous roles in a spectrum of biological contexts like maintenance of resting membrane potential , facilitation of glutamate uptake , potassium siphoning by glial cells [27, 28], cell volume and peak strength regulation of motor neurons , axonal integrity through myelination by oligodendrocytes [6, 7, 29], and cell migration . How Kir4.1 drives specific downstream signaling during disease manifestation in SeSAME syndrome requires us to understand the plethora of modifiers. Moreover, the activation of Kir4.1 depends inherently on factors like cellular milieu, the presence of auxiliary subunits, and formation of subunits for heterooligomeric assembly in cell type of choice . To address these issues and to identify other genetic associative elements with KCNJ10-mediated SeSAME pathogenesis, we performed whole-exome sequencing and functional characterization of pathogenic KCNJ10 variant in patient-specific lymphoblastoid cells which harbors the spectrum of risk variants.
Whole exome sequencing analysis of four patients and two unaffected parents identified a novel missense mutation in KCNJ10, a candidate gene in SeSAME syndrome. In addition, using two independent variant prioritization pipelines, we isolated variants in other minor genes which are known to be involved in pathways that regulate Kir4.1 signaling in different biological contexts. Along with KCNJ10, our pipeline also identified novel variants in the following genes; PIK4B (PIP2 signaling), PVRL4 (cell adhesion signaling), HSPA6 (ER-protein trafficking), and NIT1 (apoptosis). Finally, we validated the impact of KCNJ10 variant in inward-rectification of K+ current using patient-specific LCLs. The variant is localized in a stretch of conserved residues required for PIP2 binding which is juxtaposed at the junction of the transmembrane and cytoplasmic domain. Functionally, the variant alters its protein localization, accumulates in the cytoplasm, depolarizes the membranes, and inhibits inward-rectification of K+ currents in patient LCLs.
Materials and methods
Patient recruitment, genomic DNA isolation, and generation of lymphoblastoid cells
Blood samples collected from 10 participants [unaffected parents, (n = 4), and affected offsprings, (n = 6)]after receipt of informed consent were recruited at the National Institute of Mental Health and Neurosciences under aseptic conditions following guidelines established by Institutional Human Ethics Committee (IHEC) and Institutional Stem Cell committee (ISCC). The participants were referred for biochemical evaluation and selected for further analysis by presence of clinical features like seizures, ataxia, mental retardation, and hearing impairment. Genomic DNA was isolated from blood samples of all participants using NucleoSpin® Blood L (Macherey-Nagel GmbH & Co. KG) for whole-exome sequencing (WES). Peripheral blood mononuclear cells (PBMNCs) was isolated from whole blood of ten individuals and transformed by Epstein–Barr virus (EBV) using standard protocol  to generate lymphoblastoid cell lines (LCLs). The six LCLs suspensions were cultured in medium supplemented with RPMI-1640 (HiMedia AL060A), 20% fetal bovine serum (Thermo Fisher Scientific 16000-044), 1% penicillin/streptomycin (Thermo Fisher Scientific 15140-122), and maintained at 37 °C with 5% CO2 in a humidified atmosphere. The LCLs were further screened for karyotype abnormalities using G-banding approach and sample identity confirmation was done by STR profiling [GenePrint® 10 System (Promega)].
Whole exome sequencing, variant calling, quality check, and annotation
DNA library was prepared using Nextera Rapid Capture and Expanded Exome Kits. The library was further subjected to WES, performed on Illumina Hi-Sequencer to generate pair-end reads (150bp*2). We followed a whole-exome sequence analysis pipeline used by . FastQC (v0.11.5) (http://www.bioinformatics.babraham.ac.uk/projects/fastqc) was used for the quality of raw reads, which examine per base and per sequence quality scores, per base and per sequence GC content, per base N content and sequence length distribution. Prinseq-lite-0.20.4 tool was used to trim poor quality region (http://prinseq.sourceforge.net/) and adapterremoval-2.1.7 was used to remove adapter contamination in raw reads. Filtered reads with a quality score (Q) > 20 were aligned to the human reference genome hg19 (GRCh37) using BWA (v0.5.9). SAM to BAM conversion and sorting were done with Samtools 1.3 tool (https://sourceforge.net/projects/samtools/files/samtools/1.3/). Then the PCR duplicates were removed using PICARD tools (v1.96) (https://broadinstitute.github.io/picard/) and the INDELS were realigned using GATK (v3.6). The BAM alignment was subjected to QC using Qualimap (v2.2). VarScan (v2.3.9) (coverage = 8, MAF > = 0.25, p value < 0.001) was used to call for SNPs and INDELS. The quality of VCF file was checked using RTG tools 3.7.1 (https://github.com/RealTimeGenomics/rtg-tools/releases). All samples annotation was performed using ANNOVAR tool. Population controls (n = 7) representing three religious groups (group A, B, and C) matched for age, sex, and ethnicity, were obtained from INDEX-db . All controls passed the age of risk i.e., 45 years, for neuropsychiatric illnesses, except for the outbred Parsi (religious group 3) individual (age = 26), who was included as an outlier. All the controls were of southern Indian ethnic origin except for the Parsi. To validate KCNJ10 variant identified by whole-exome sequencing, we performed Sanger validation using the following gene-specific primers: Forward (CATTCGTTTCAGCCAGCATGC) and Reverse (TCAGACATTGCTGATGCGCA).
Assessing runs of homozygosity
Exome-wide F-statistics was calculated using the --het option in vcftools (v0.1.5), for every sample to investigate whether levels of heterozygosity differed between the affected siblings, unaffected parents and population controls. Runs of homozygosity (ROH) was detected in all samples using --homozyg option in PLINK (v1.9) . The minimum length for a tract to qualify as ROH was set to 500kb and the minimum number of variants constituting an ROH was set to 100. A maximum of three intervening heterozygous variants were allowed within a ROH window. ROH density was set to default, i.e., an ROH must have at least one variant per 50kb, on an average. The centromeric, X, Y and mitochondrial variants were ignored during this analysis. The stretches that were shared between all the affected individuals but not observed in either of the parents or the population controls were thus notified as ROHaffected, which were identified by using a combination of intersect and subtract functions in bedtools (v2.22). The variants were annotated using variant effect predictor (VEP GRCh37).
Whole-cell patch-clamp electrophysiology
For electrophysiology studies, LCLs from a healthy wild type control, six participants from SeSAME like family described in this study were used. The LCLs were dissociated to single cells and plated on glass coverslips coated with poly-D-lysine (Millipore, A003M EMD) and incubated for half an hour at 37 °C with 5% CO2 in a humidified atmosphere before recordings. Whole cell patch-clamp recordings were configured following which the membrane potential (Vm) of LCLs was measured. A pulse protocol was applied with Vm held at resting membrane potential and then stepped to test potentials between − 120 mV and 40 mV in 10 mV steps for 140 ms. A single electrode was used to measure membrane current (nA) by whole-cell patch-clamp technique. Intracellular voltage-clamp recordings and positioning of perfusion micropipette were done using two Narashige hydraulic micromanipulators (MNW-203, Narashige, Japan). Recording pipettes (tip resistance 4–6 MΩ) were filled with intracellular solution containing 120 mM potassium D-gluconate (G4500, Sigma), 1 mM MgCl2, 15 mM KCl, 1 mM CaCl2, 10 mM EGTA, and 10 mM HEPES (pH7.2). After obtaining whole-cell mode, access resistance was 10–15 MΩ. The extracellular recording solution contained 130 mM NaCl, 3 mM CaCl2, 2.5 mM MgCl2, 15 mM HEPES (pH7.4). In experiments, where LCLs were perfused with high extracellular K+, concentration of KCl varied from 5–20 mM while that of NaCl was decreased to 110 mM to adjust osmolarity. Recordings in LCLs were performed using a HEKA triple patch-clamp amplifiers (EPC 10 USB) at room temperature (RT). To determine specificity of Kir4.1 current, 110 μm/L BaCl2 was used and to block endogenous Cl- currents, 150 μm/L niflumic acid was used in the bath solution. The pClamp 9 (Axon Instruments) software package was used for data acquisition and analysis. For statistical analysis, we used GraphPad Prism (San Diego, USA). To choose between parametric or non-parametric tests for normality criteria, Shapiro-Wilk estimator was used. For data sets with small N, non-parametric test was used to avoid possible type II errors. Mean differences were statistically evaluated using ANOVA with Levene’s homogeneity of variances test and pairwise comparisons were made using Turkey adjustment. Non-parametric k independent Kruskal-Wallis test was applied with Bonferroni correction to compare the differences among means. Error bars represent +S.E.
Immunofluorescence and western blotting
The LCLs were fixed using 4% paraformaldehyde (Sigma, PFA: P6148) in phosphate-buffered saline (PBS) for 20 min at RT. Cells were permeabilized using 0.2% Triton X-100 (Sigma, T8787) for 10 min and were washed twice with PBS. Following permeabilization cells were blocked for 1 h using 2% bovine serum albumin (BSA) in PBST (PBS containing 0.05% tween 20; Sigma, P2287). Primary antibody against hKir 4.1 (1:100, Novus biologicals, NBP1-20149) was incubated overnight at 4 oC in block solution. Cells were washed twice with PBST followed by 1 h incubation at RT with anti-rabbit Alexa FluorTM 488 (1:200; Thermo Fisher Scientific, A11001) and Alexa FluorTM 568 phalloidin (1:200; Thermo Fisher Scientific, A12380). Following incubation cells were washed twice with PBST and incubated with DAPI (1:10000; Thermo Fisher Scientific, 62248) for 10 min at RT. The cells were washed twice with PBS and mounted using Vectashield antifade mounting medium (H-1000: Vector labs). Optical z-sectioning at 0.2 μM intervals was done using Plan-Apochromat 63x/1.40 oil objective in Zeiss Axio Observer 7 with Apotome 2 feature and Axiocam 702 monochrome camera (Carl Zeiss, Germany). Signal-to-noise ratio was improved using the in-built Zeiss deconvolution module and MIP projections of 2–3 Z-stacks are presented here. Representative images reported here are from three independent experiments. For quantitative measurements, deconvoluted Z-stacks were first blinded before analysis. 3D surface rendering plugin in Imaris software is used to reduce signal-noise ratio to measure Kir4.1 punctate distribution between cytoplasm and nucleus. The respective numbers were normalized against cytoplasmic space marked by F-actin and nuclear space by DAPI signals.
LCLs suspension of all six participants was cleared by centrifugation (1500 rpm for 3 min) to remove culture media. RIPA lysis buffer containing phosphatase and protease inhibitor cocktails (EDTA-free, ab201120) was used to lyse the cells and total protein was isolated. Bradford assay was used to measure the concentration of the protein. All six samples (20 ug protein /lane) were resolved using 10% SDS-PAGE, transferred to PVDF membrane and probed with anti-Kir4.1 protein (NBP1-20149) and β-actin (A5441) as loading control. Target protein bands detection was done in Gel Documentation system (Syngene: chemiXX9) using Super signal West Pico Chemiluminescent substrate (Thermo Scientific, #34077) and densitometric quantitation assessed using Image Studio Lite v5.2 (LI-COR Biosciences).
Clinical features of a family with SeSAME syndrome
Six affected patients, born through two consanguineous unions, were identified from the relatives of an index patient (IV.2) who developed tonic-clonic seizures, ataxia, and developmental delay (Fig. 1a). The clinical features were broadly similar to SeSAME syndrome but without electrolyte imbalance (Table 1). The cerebellar symptoms (gait ataxia, intentional tremors, and dysdiadochokinesia) were manifested from early childhood. The gait ataxia was progressive in nature, resulting in severe disability and later being confined to wheelchairs [IV. 2–5]. Dysmorphic facies, dysarthria, brisk deep tendon reflexes (DTRs), bilateral ankle clonus, and an extensor Babinski response were evident in all of them. All the patients showed certain characteristic dysmorphic facial features like prominent supraorbital ridges, thick eyebrows, deep-set eyes, epicanthal fold, low set ears, prominent antihelix, prominent nasal tip, and thick lips (Fig. 1b). Behavioral abnormalities like stereotypies, hyperactivity, anger outbursts, and psychotic symptoms were also observed (Table 1). They also had hearing impairment, and audiometry measures revealed bilateral mild to severe sensory neural hearing loss. Motor nerve conduction velocities from patients (V.1–2) were normal. The EEG from patients (V.1–2) showed generalized seizure discharges before treatment (Fig. 1c), which became normal after treatment with anti-epileptic drugs. The other four members (IV.2–5) remained seizure-free for several years on medication. MRI from IV.2 showed enlarged basal ganglia and cerebellar atrophy (Fig. 1d). The remaining members of the family were clinically unaffected.
Variant prioritization using ROH and non-ROH methods identified two novel variants in KCNJ10 and PI4KB and revealed mutation burden in Chr 1 in all patients
To identify the critical disease-associated loci, we performed WES and prioritized variants based on two independent approaches; assessing the exome-wide levels of homozygosity (ROH method) and assessing variants based on allele frequencies with autosomal recessive inheritance pattern (non-ROH method) in all family members. Unanimously, both analysis pipelines identified two novel high-risk disease-associated variants in KCNJ10 and PI4KB and five rare variants in PVRL4, RORC, FLG2, FCRL1, and NIT1 and one common variant in HSPA6 segregating in homozygous state in all patients and heterozygous state in both parents. Surprisingly, both methods revealed mutational burden in Chr1 (Fig. 2a; Table 2).
Deleterious genetic effects of inbreeding are evident in children born out of consanguineous unions with a relatively higher burden of homozygous alleles [34,35,36]. These effects have been implicated to influence the evolution of mental illness and neurodevelopmental disorders . Since SeSAME syndrome follows autosomal recessive (AR) inheritance and the role of homozygous alleles in AR illness has been well established , we analyzed the exome-wide levels of homozygosity for all samples within the pedigree including seven population controls (see “Materials and methods” section). Principal component analysis (PCA) of the exome-wide F-statistics separates the family members (n = 6) from the population controls (n = 7), explaining for an overall variance of 49.6%. All samples (both familial and population) within the two clusters, fell within their 95% confidence ellipses, except for two controls representing the relatively admixed communities (Fig. 2b). The SeSAME family alone was subjected to PCA in which the cases (n = 4) formed a cluster and the unaffected parents (n = 2) fell outside the 95% confidence ellipse (Fig. 2c), explaining the intra-familial variance in homozygosity. The ROH within the exomes of the individuals in the pedigree and the population controls were identified. A total of 56 homozygous stretches (either overlapping or unique) were identified in all cases and controls, of which 44 stretches belonged to the 4 affected siblings and the remaining were distributed between unaffected parents and population controls (Additional file 1: Table S1). Nevertheless, no ROH was detected in a subset of population controls. The burden of ROHs witnessed in the cases as compared to controls could be attributed to their consanguineous parentage. Of the ROHs identified in total, five stretches were explicitly shared between all the affected siblings but not observed in the unaffected parents and population controls, which will henceforth be notified as ROHaffected (Fig. 2d). TheROHaffected consists of a union set of 5329 variants across all the cases and controls, of which any given variant was observed in at least one sample. Since the disorder follows an autosomal recessive (AR) inheritance pattern, of the 5329 variants, we identified those that were heterozygous (HET) in both unaffected parents, but homozygous (HOM) in all of the affected siblings. Seventy-eight such variants, belonging to 47 genes, were identified, and all of them mapped to Chr 1 (Additional file 1: Table S2). This skewed observation could not be attributed to the length of Chr 1 for three reasons: (i) the method used to compute ROH uses a sliding window approach which essentially removes the bias induced by the length of the chromosome; (ii) the same Chr 1 ROH was not observed in either of the controls; (iii) no ROH was observed in Chr 2 despite its genomic length being comparable to that of Chr 1. Of the 78 variants, only 3 missense variants, i.e., (i) Chr1:158368964-C-T (OR10T2), (ii) Chr1:160011455-T-C (KCNJ10), and (iii) Chr1:161495040-C-T (HSPA6), were predicted to be deleterious by two algorithms.
To identify other deleterious variants segregating within the family by AR pattern, which could have otherwise been ignored by the ROH based method, we identified all the exonic and splice variants (including non-synonymous, stop gain, and stop loss). The common variants, i.e., those with a minor allele frequency (MAF) > 0.01 in 1 kg_all (1000 Genomes Project) and ExAC_all (Exome Aggregation Consortium) databases, were excluded from the analysis. We identified seven variants belonging to seven genes (Additional file 1: Table S3). Interestingly, all seven variants were located within Chr1:151288779-161088292, which was a subset of ROHaffected (Fig. 2d). Among the seven variants, Chr1:160011455-T-C [KCNJ10] was an obvious overlap. The remaining six variants fell on PI4KB, RORC, FLG2, FCRL1, PVRL4, and NIT1 genes. Apart from KCNJ10 variant, none were predicted to be deleterious by all six prediction algorithms. However, three of the remaining six variants (Chr1:151288779-T-C [PI4KB], Chr1:161049499-G-A [PVRL4], and Chr1:161088292-A-G [NIT1]) were predicted to be deleterious by at least two algorithms (Table 2). Finally, the zygosity of the KCNJ10 variant was confirmed by sanger sequencing for six patients and four unaffected parents in the family (III.11–12, IV.2–5, IV.9–10, and V.1–2) (Fig. 2e).
Thus, of the union set of nine putative deleterious variants (three based on ROH method and seven based on allele frequencies) segregating within the family, the KCNJ10 gene was shortlisted for functional analysis to unravel the molecular impact of the variant for following reasons: (i) KCNJ10, the candidate gene known to cause SeSAME syndrome (Celmina et al. 2018); (ii) the variant reported in the patients is novel; (iii) this was the only deleterious variant identified by both methods and (iv) the variant resides at the interface between transmembrane and cytoplasmic domain at the membrane (Fig. 2f) which is strongly conserved through evolution (Fig. 2g).
Novel KCNJ10 variant disrupts channel properties in patient-derived LCLs
LCLs have been routinely used as a surrogate in vitro cell model to investigate cellular mechanisms of neurodevelopmental psychiatric disorders . To investigate the functional role of Kir4.1T290A, we generated patient-specific LCLs, validated by karyotype for six members of SeSAME family. All six LCLs are free from both numeric and structural chromosomal abnormalities (data not shown).
The barium-sensitive inwardly-rectifying K+ current in LCLs measured by whole-cell patch-clamp was substantially compromised in all patients. Kir4.1T290A significantly depolarized LCL membranes and showed deficits in clearance of extracellular K+. To determine whether LCLs express functionally active endogenous Kir4.1 protein, we used immunofluorescence (IF), western blot, and electrophysiology (Fig. 3). In parental controls, Kir4.1 is in close proximity with the actin-rich plasma membrane, diffusely discernible in the cytoplasm and enriched in the nuclear membrane and nucleus (Fig. 3a). However, in all affected individuals, we observed an increased punctate distribution of Kir4.1 in the cytoplasm but with no apparent disparity in the nucleus and nuclear membrane (Fig. 3b). To confirm the IF findings, western blot analysis showed a substantial increase in the expression of Kir4.1 in all patients compared with unaffected parents (Fig. 3c and d). These findings suggest an unstable nature of the mutant Kir4.1T290A in all patients.
To confirm whether the endogenous Kir4.1 expressed in LCLs is functionally active and elicit detectable inward-rectifying potassium currents in vitro, we performed whole-cell patch-clamp recordings in response to voltage-steps from − 120 to 40 mV in 10 mV, from a holding potential of − 30 mV both in the presence and absence of 110 μM barium, a selective Kir channel blocker. Baseline current discharges from two heterozygous parental controls (III.11 − 0.89 + 0.086, n = 18, p = 1.114 and III.12 − 0.86 + 0.049, n = 16, p = 1.347) were not significantly different from wild type controls (− 0.85 + 0.046, n = 17) (Fig. 3e). In contrast, the average barium-sensitive current densities were substantially decreased in all three control LCLs tested, in heterozygous parents (III.11 − 0.64 + 0.041, n = 15, p = 2.1E−4 and III.12 − 0.60 + 0.086, n = 14, p = 1.8E−4) and wild type (− 0.63 + 0.104, n = 14, p = 2.5E−4) compared with their respective baseline discharges, implying the specificity of K+ currents recorded from endogenous Kir channels (Fig. 3e).
We recorded the resting membrane potential of LCLs from patients (Fig. 3f). Average membranes voltages from all patients (IV.2 − 30 mV + 3.640, n = 18, p = 1.3E−5; IV.3 −32 mV + 2.156, n = 20, p = 2.4E−5; IV.4 − 31 mV + 3.083, n = 17, p = 1.7E−4; IV.5 − 24 mV + 2.817, n = 20, p = 2.8E−5) were significantly hyperpolarized as compared to wild type (WT −55 mV + 4.102, n = 24) and parental controls (III.11 − 51 mV + 3.842, n = 21 and III.12 − 50 mV + 4.21, n = 19). In whole-cell voltage-clamp, membrane current amplitudes were measured in all family members at both positive and negative potentials than the K+ equilibrium potential (Ek) (Fig. 3g and h). The mean current densities as a function of voltage (pA/pF) measured in all those expressing the mutant channel were markedly smaller than wild type and parental controls (Fig. 3h). One major facet of the Kir4.1 channel is to clear extracellular K+ thereby showing stronger rectification. To test the K+ clearance ability of LCLs, we clamped the cells at their resting membrane potential, with and without 110 μM barium, and measured the elicited membrane current discharges upon induced K+steps (from 5 to 20 mM). Overall, barium-sensitive currents from all patients were significantly reduced when compared to both parental and wild type controls (Fig. 3i).
In this study, we identified two novel pathogenic variants in KCNJ10 and PI4KB, five rare pathogenic variants in PVRL4, RORC, FLG2, FCRL1, and NIT 1 and one common pathogenic variant in HSPA6 suggesting the importance of membrane lipid signaling, adhesion-mediated cell migration, and protein trafficking in SeSAME syndrome through regulation of Kir channel activity. In multiple biological contexts, these cellular processes are tightly linked in regulating Kir4.1 channel function at the plasma membrane [9, 39,40,41,42,43]. Functional studies in patient-specific LCLs suggests that the variant in KCNJ10 causes 60% reduction in Kir4.1 channel activity which is presumably due to altered protein localization and decreased surface expression of mutant proteins. Finally, our study identified risk-associated variants in seven new genes in SeSAME syndrome, which might act as modifiers by regulating Kir4.1 channel function. A detailed mechanistic study investigating the biology of these modifiers in Kir4.1 physiology will help us to underpin the biology of disease manifestation in SeSAME syndrome.
Signal-dependent Golgi export processes have been implicated in Andersen-Tawil syndrome (ATS1) by controlling the surface density Kir2.1 channel . It has become evident in recent years that differential trafficking of Kir channels controls neuronal excitability, hormone secretion, action potential, K+ homeostasis, and salt balance. The shared Golgi export signal patch at the cytoplasmic region in Kir2.3 and Kir4.1 is an AP-1 clathrin adaptor recognition site which ensures an additional quality control checkpoint for the exit of mature folded channels . The variant reported in this study Kir4.1T290A, reside in close proximity to Golgi export patch at the cytoplasmic region, implying the role of protein trafficking in SeSAME syndrome. Supporting this view, non-ROH method of analysis identified a pathogenic common variant in HSPA6 gene, a molecular chaperone involved in ATP-dependent protein quality control system. It is also interesting to note the association of HSPA6 variant in patients with sensory disturbances  suggesting mutations in genes that regulate protein trafficking can influence surface expression of Kir4.1 channel, irrespective of its variants.
All six patients reported here displayed relatively uniform and expected neurological and psychiatric manifestations, but they did not manifest electrolyte imbalance. Therefore, how and why certain KCNJ10 variants fail to manifest electrolyte imbalance in SeSAME syndrome needs to be explored. There could be two possibilities for this discrepancy. First, it is possible that certain KCNJ10 mutations can affect CNS functions independently of other organ systems. It is conceivable that astrocytes and microglial cells of the nervous system are highly sensitive to dysregulation of potassium homeostasis, while basolateral membrane in the distal nephron may be impervious to this effect . Another possibility is that same KCNJ10 variants could behave differently between CNS and kidney, since the channel activity depends largely on the formation of heterotetramers with other Kir entities (Kir5.1), cell-type specificity, gating mechanisms, and its influence on cell surface signaling receptors through PIP2 binding [9, 40, 43, 46]. In addition, it is unclear whether renal electrolyte deficit is a progressive impairment that develops over time, or a direct effect of the mutation, which necessitates further investigations and follow-up clinical evaluations. These different mechanisms suggest that although major gene effects are probably the primary drivers of illness, the diversity in clinical presentation is perhaps an outcome of complex genetic interactions between common and rare variants, each of varying effect sizes.
Surprisingly, both methods concluded a mutational and ROH burden in Chr 1. Given the clinical diversity and for additional reasons as discussed above, we suggest two possibilities for ROH and mutational burden which are broadly classified into intrinsic and extrinsic factors. Intrinsic factors include recombination hot-spots, defects in DNA repair, chromatin remodeling, and yet unidentified intracellular signaling events, that favor to the occurrence of ROH, co-segregating with the illness. The extrinsic factor could be the clan structure of the family, which indicates a high degree of endogamy. Another possibility is that individual ROHs might play key role in spatial-temporal regulation of gene expression within cell types that are sensitive to K+ homeostasis. The difference in the expression of Kir4.1 in patients in our SeSAME pedigree also highlights the role of ROH in gene regulation. Therefore, it would be helpful to investigate the functional consequences of homozygosity in expression of genes within the ROH and/or in close proximity especially in cell types that are relevant to the pathophysiology of SeSAME syndrome. Finally, an interplay between these factors could help us discriminate the cause and effect relationship of ROH in clinical diversity of SeSAME syndrome. Usually for every pregnancy in autosomal recessive disorders, there is a probability of 0.25 that the offspring(s) will inherit two copies of the disease gene and will therefore exhibit the phenotype . However, in a clinical setting this distribution is skewed more towards almost all affected individuals in the same generation, than one would rather expect by chance, especially in children born to consanguineous unions. Thus, this skewed observation needs to be addressed at holistic paradigms by developing biophysical and mathematical models to understand the physics and governing dynamics of the intracellular events, influencing the silent recombination choices of homologous chromosomes.
Though our study identified novel and common variants in new genes and its pathways that could help modify the activity of Kir channels in SeSAME pathogenesis, a complete mechanistic understanding would require the establishment of animal models to explore the cell-type-specific role of Kir4.1 in brain function. Justifying the importance of K+ homeostasis in the brain, Kir4.1 knockout mouse, Xenopus, zebrafish, and Drosophila mimic a subset of SeSAME symptoms in humans [6, 7, 10, 26, 29, 48, 49]. Therefore, future experiments with in vivo model systems will help dissect the cross-talk of Kir4.1 signaling with membrane lipids , cell adhesion in axon guidance and synaptic architecture which is an essential feature for proper synaptic transmission and plasticity.
Our study identified two novel and five rare variants in genes that potentially modify the channel properties of Kir4.1-mediated pathogenesis in SeSAME syndrome. In the future, genetic interaction experiments in cell and/or animal model systems will help us tease apart the causative effects of these novel modifiers in Kir4.1 biology. Only a subset of reported variants in Kir4.1 gene exhibited strong functional defects in inward rectification, which makes it difficult explain the causative nature of complex SeSAME syndrome by one major gene effect. Our study provides evidence for the role of minor genes as well from different signaling pathways which might either contribute or influence Kir4.1-mediated disease pathology.
Availability of data and materials
The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.
Bovine serum albumin
Epilepsy, Ataxia, Sensorineural deafness, Tubulopathy
Peripheral blood mononuclear cells
Principal component analysis
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The authors are immensely grateful to all members of SeSAME family for their participation and constant involvement in this study. We thank Dr. Gautham Arunachal U (NIMHANS) for describing the various dysmorphic features of the SeSAME kindreds. We thank ADBS genomic team for sharing the WES analysis pipeline. This work was generously supported with funds from Ramalingaswami re-entry fellowship (RLF/DBT/2015), ADBS (BT/PR17316/MED/31/326/2015) from the Department of Biotechnology and Department of Science and Technology (ECR/2015/000468). Finally, the authors are thankful to all members of the molecular genetics, ADBS lab and the ADBS consortium for suggestions and discussions during the course of investigation.
These experiments were supported by Ramalingaswami re-entry fellowship (RLF/DBT/2015) and ADBS (BT/PR17316/MED/31/326/2015) from Department of Biotechnology; early career grant (ECR/2015/000468) from Department of Science and Technology.
Ethics approval and consent to participate
The study has been approved by three institutional committees; NIMHANS ethics committee [NIMHANS/2nd IEC (BS and NS DIV)/2016], NIMHANS stem cell research committee (IC-SCR) dated 12 February 2018 and NIMHANS bio-safety committee (NIMHANS/DO/3rd IBSC MEET1NG/2016).
Consent for publication
We have obtained the consent for publication from all individuals. We would be glad to share this data at any stage when requested.
The authors declare that they have no competing interests.
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Table S1. ROH (n=56) (either overlapping or unique) detected within the exomes of all cases and controls. No ROH detected in the populations controls 7, 8 and 9. Table S2. Variants (n=78) within ROHpro that were HET in all of the unaffected parents and HOM in all of the affected siblings. (0/1 = HET; 1/1 = HOM). Table S2. Variants (n=78) within ROHpro that were HET in all of the unaffected parents and HOM in all of the affected siblings. (0/1 = HET; 1/1 = HOM). Table S2. Variants (n=78) within ROHpro that were HET in all of the unaffected parents and HOM in all of the affected siblings. (0/1 = HET; 1/1 = HOM). Table S2. Variants (n=78) within ROHpro that were HET in all of the unaffected parents and HOM in all of the affected siblings. (0/1 = HET; 1/1 = HOM). Table S2. Variants (n=78) within ROHpro that were HET in all of the unaffected parents and HOM in all of the affected siblings. (0/1 = HET; 1/1 = HOM). Table S3. Variants (n=7) shortlisted based on assessing the allele frequencies (MAF<0.01) in 1KG_all and ExAC_all. The below variants are the only those which were HET in all unaffected parents and HOM in all affected siblings. (XLSX 31 kb)
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Nadella, R.K., Chellappa, A., Subramaniam, A.G. et al. Identification and functional characterization of two novel mutations in KCNJ10 and PI4KB in SeSAME syndrome without electrolyte imbalance. Hum Genomics 13, 53 (2019) doi:10.1186/s40246-019-0236-0