Mutations In SLC9A6 Research Articles

Targeting NHE6 gene expression identifies lysosome and neurodevelopmental mechanisms in a haploid in vitro cell model.

Christianson syndrome (CS) is an X-linked disorder resulting from loss-of-function (LoF) mutations in SLC9A6 encoding the endosomal Na+/H+ exchanger 6 (NHE6). CS presents with developmental delay, seizures, intellectual disability, nonverbal status, postnatal microcephaly, and ataxia. To define transcriptome signatures of NHE6 LoF, we conducted in-depth RNA-sequencing (RNA-seq) analysis on a haploid NHE6 null cell model. CRIPSR/Cas9 genome editing introduced multiple LoF mutations into SLC9A6 in the near haploid human cell line Hap1. Isogenic, paired parental controls were also studied. NHE6 mutant cell lines were confirmed to have intra-endosomal over-acidification as was seen in other NHE6 null cells. RNA-seq analysis was performed by two widely used pipelines: HISAT2-StringTie-DEseq2 and STAR-HTseq-DEseq2. We identified 1056 differentially expressed genes in mutant NHE6 lines, including genes associated with neurodevelopment, synapse function, voltage-dependent calcium channels, and neuronal signaling. Weighted gene co-expression network analysis was then applied and identified a critical module enriched for genes governing lysosome function. By identifying significantly changed gene expression that is associated with lysosomal mechanisms in NHE6-null cells, our analyses suggest that loss of NHE6 function may converge on mechanisms implicated in lysosome-related neurologic disease. Further, this haploid cell model will serve as an important tool for translational science in CS.

Structural and functional implications of SLC13A3 and SLC9A6 mutations: an in silico approach to understanding intellectual disability

BackgroundIntellectual disability (ID) is a condition that varies widely in both its clinical presentation and its genetic underpinnings. It significantly impacts patients’ learning capacities and lowers their IQ below 70. The solute carrier (SLC) family is the most abundant class of transmembrane transporters and is responsible for the translocation of various substances across cell membranes, including nutrients, ions, metabolites, and medicines. The SLC13A3 gene encodes a plasma membrane-localized Na+/dicarboxylate cotransporter 3 (NaDC3) primarily expressed in the kidney, astrocytes, and the choroid plexus. In addition to three Na + ions, it brings four to six carbon dicarboxylates into the cytosol. Recently, it was discovered that patients with acute reversible leukoencephalopathy and a-ketoglutarate accumulation (ARLIAK) carry pathogenic mutations in the SLC13A3 gene, and the X-linked neurodevelopmental condition Christianson Syndrome is caused by mutations in the SLC9A6 gene, which encodes the recycling endosomal alkali cation/proton exchanger NHE6, also called sodium-hydrogen exchanger-6. As a result, there are severe impairments in the patient’s mental capacity, physical skills, and adaptive behavior.Methods and resultsTwo Pakistani families (A and B) with autosomal recessive and X-linked intellectual disorders were clinically evaluated, and two novel disease-causing variants in the SLC13A3 gene (NM 022829.5) and the SLC9A6 gene (NM 001042537.2) were identified using whole exome sequencing. Family-A segregated a novel homozygous missense variant (c.1478 C > T; p. Pro493Leu) in the exon-11 of the SLC13A3 gene. At the same time, family-B segregated a novel missense variant (c.1342G > A; p.Gly448Arg) in the exon-10 of the SLC9A6 gene. By integrating computational approaches, our findings provided insights into the molecular mechanisms underlying the development of ID in individuals with SLC13A3 and SLC9A6 mutations.ConclusionWe have utilized in-silico tools in the current study to examine the deleterious effects of the identified variants, which carry the potential to understand the genotype-phenotype relationships in neurodevelopmental disorders.

Functional analysis of two SLC9A6 frameshift variants in lymphoblastoid cells from patients with Christianson syndrome.

Christianson syndrome (CS) is caused by mutations in SLC9A6 and is characterized by global developmental delay, epilepsy, hyperkinesis, ataxia, microcephaly, and behavioral disorder. However, the molecular mechanism by which these SLC9A6 mutations cause CS in humans is not entirely understood, and there is no objective method to determine the pathogenicity of single SLC9A6 variants. Trio-based whole exome sequencing (WES) was carried out on two individuals with suspicion of CS. qRT-PCR, western blot analysis, filipin staining, lysosomal enzymatic assays, and electron microscopy examination, using EBV-LCLs established from the two patients, were performed. Trio-based WES identified a hemizygous SLC9A6 c.1560dupT, p.T521Yfs*23 variant in proband 1 and a hemizygous SLC9A6 c.608delA, p.H203Lfs*10 variant in proband 2. Both children exhibited typical phenotypes associated with CS. Expression analysis in EBV-LCLs derived from the two patients showed a significant decrease in mRNA levels and no detectable normal NHE6 protein. EBV-LCLs showed a statistically significant increase in unesterified cholesterol in patient 1, but only non-significant increase in patient 2 when stained with filipin. Activities of lysosomal enzymes (β-hexosaminidase A, β-hexosaminidase A + B, β-galactosidase, galactocerebrosidase, arylsulfatase A) of EBV-LCLs did not significantly differ between the two patients and six controls. Importantly, by electron microscopy we detected an accumulation of lamellated membrane structures, deformed mitochondria, and lipid droplets in the patients' EBV-LCLs. The SLC9A6 p.T521Yfs*23 and p.H203Lfs*10 variants in our patients result in loss of NHE6. Alterations of mitochondria and lipid metabolism may play a role in the pathogenesis of CS. Moreover, the combination of filipin staining with electron microscopy examination of patient lymphoblastoid cells can serve as a useful complementary diagnostic method for CS.

Deletion of the sodium/hydrogen exchanger 6 causes low bone volume in adult mice.

The sodium/hydrogen exchanger 6 (NHE6) localizes to recycling endosomes, where it mediates endosomal alkalinization through K+/H+ exchange. Mutations in the SLC9A6 gene encoding NHE6 cause severe X-linked mental retardation, epilepsy, autism and corticobasal degeneration in humans. Patients with SLC9A6 mutations exhibit skeletal malformations, and a previous study suggested a key role of NHE6 in osteoblast-mediated mineralization. The goal of this study was to explore the role of NHE6 in bone homeostasis. To this end, we studied the bone phenotype of NHE6 knock-out mice by microcomputed tomography, quantitative histomorphometry and complementary ex vivo and in vitro studies. We detected NHE6 transcript and protein in both differentiated osteoclasts and mineralizing osteoblasts. In vitro studies with osteoclasts and osteoblasts derived from NHE6 knock-out mice demonstrated normal osteoclast differentiation and osteoblast proliferation without an impairment in mineralization capacity. Microcomputed tomography and bone histomorphometry studies showed a significantly reduced bone volume and trabecular number as well as an increased trabecular space at lumbar vertebrae of 6months old NHE6 knock-out mice. The bone degradation marker c-terminal telopeptides of type I collagen was unaltered in NHE6 knock-out mice. However, we observed a reduction of the bone formation marker procollagen type 1 N-terminal propeptide, and increased circulating sclerostin levels in NHE6 knock-out mice. Subsequent studies revealed a significant upregulation of sclerostin transcript expression in both primary calvarial cultures and femora derived from NHE6 knock-out mice. Thus, loss of NHE6 in mice causes an increase of sclerostin expression associated with reduced bone formation and low bone volume.

Functional Assessment In Vivo of the Mouse Homolog of the Human Ala-9-Ser NHE6 Variant.

Christianson syndrome (CS) is an X-linked neurogenetic disorder resulting from loss-of-function (LoF) mutations in SLC9A6, which encodes the endosomal Na+/H+ exchanger 6 (NHE6). NHE6 regulates proton efflux from endosomes and, thus, participates in regulating cargo processing and trafficking. LoF mutations in NHE6 cause aberrant acidification of endosomes. While CS arises in males generally due to clear LoF mutations, other potentially hypomorphic variants have emerged, yet most of these variants have not been evaluated for functional effects, particularly in vivo. Here we characterize an SLC9A6 variant that has been previously reported in patients, yet now also appears in exome datasets of largely control individuals—c.25G>T, p.A9S. By heterologous expression in cell lines, we show that human NHE6A9S is expressed and localizes in a manner comparable to control NHE6. By genome editing, we generated the equivalent NHE6 mutation in mouse—p.A11S—and determined that male NHE6A11S mice have normal brain size at 6 months of age and do not show cerebellar degeneration or defective neuronal arborization. Neurons from male NHE6A11S mice also did not demonstrate an abnormality in intraendosomal pH compared with controls. These findings are in contrast to findings in NHE6-null mice previously reported and indicate that the NHE6A11S variant functions at a level equivalent to control NHE6 for many of the assays performed. These data stand in support of the population genetic data, which are also evaluated here, indicating that the A9S variant is unlikely to confer disease susceptibility with high penetrance.

Pathobiology of Christianson syndrome: Linking disrupted endosomal-lysosomal function with intellectual disability and sensory impairments.

Christianson syndrome (CS) is a recently described rare neurogenetic disorder presenting early in life with a broad range of neurological symptoms, including severe intellectual disability with nonverbal status, hyperactivity, epilepsy, and progressive ataxia due to cerebellar atrophy. CS is due to loss-of-function mutations in SLC9A6, encoding NHE6, a sodium-hydrogen exchanger involved in the regulation of early endosomal pH. Here we review what is currently known about the neuropathogenesis of CS, based on insights from experimental models, which to date have focused on mechanisms that affect the CNS, specifically the brain. In addition, parental reports of sensory disturbances in their children with CS, including an apparent insensitivity to pain, led us to explore sensory function and related neuropathology in Slc9a6 KO mice. We present new data showing sensory deficits in Slc9a6 KO mice, which had reduced behavioral responses to noxious thermal and mechanical stimuli (Hargreaves and Von Frey assays, respectively) compared to wild type (WT) littermates. Immunohistochemical and ultrastructural analysis of the spinal cord and peripheral nervous system revealed intracellular accumulation of the glycosphingolipid GM2 ganglioside in KO but not WT mice. This cellular storage phenotype was most abundant in neurons of lamina I-II of the dorsal horn, a major relay site in the processing of painful stimuli. Spinal cords of KO mice also exhibited changes in astroglial and microglial populations throughout the gray matter suggestive of a neuroinflammatory process. Our findings establish the Slc9a6 KO mouse as a relevant tool for studying the sensory deficits in CS, and highlight selective vulnerabilities in relevant cell populations that may contribute to this phenotype. How NHE6 loss of function leads to such a multifaceted neurological syndrome is still undefined, and it is likely that NHE6 is involved with many cellular processes critical to normal nervous system development and function. In addition, the sensory issues exhibited by Slc9a6 KO mice, in combination with our neuropathological findings, are consistent with NHE6 loss of function impacting the entire nervous system. Sensory dysfunction in intellectually disabled individuals is challenging to assess and may impair patient safety and quality of life. Further mechanistic studies of the neurological impairments underlying CS and other genetic intellectual disability disorders must also take into account mechanisms affecting broader nervous system function in order to understand the full range of associated disabilities.

Mixed Neurodevelopmental and Neurodegenerative Pathology in Nhe6-Null Mouse Model of Christianson Syndrome.

Christianson syndrome (CS) is an X-linked disorder resulting from loss-of-function mutations in SLC9A6, which encodes the endosomal Na+/H+ exchanger 6 (NHE6). Symptoms include early developmental delay, seizures, intellectual disability, nonverbal status, autistic features, postnatal microcephaly, and progressive ataxia. Neuronal development is impaired in CS, involving defects in neuronal arborization and synaptogenesis, likely underlying diminished brain growth postnatally. In addition to neurodevelopmental defects, some reports have supported neurodegenerative pathology in CS with age. The objective of this study was to determine the nature of progressive changes in the postnatal brain in Nhe6-null mice. We examined the trajectories of brain growth and atrophy in mutant mice from birth until very old age (2 yr). We report trajectories of volume changes in the mutant that likely reflect both brain undergrowth as well as tissue loss. Reductions in volume are first apparent at 2 mo, particularly in the cerebellum, which demonstrates progressive loss of Purkinje cells (PCs). We report PC loss in two distinct Nhe6-null mouse models. More widespread reductions in tissue volumes, namely, in the hippocampus, striatum, and cortex, become apparent after 2 mo, largely reflecting delays in growth with more limited tissue losses with aging. Also, we identify pronounced glial responses, particularly in major fiber tracts such as the corpus callosum, where the density of activated astrocytes and microglia are substantially increased. The prominence of the glial response in axonal tracts suggests a primary axonopathy. Importantly, therefore, our data support both neurodevelopmental and degenerative mechanisms in the pathobiology of CS.

Christianson syndrome: An underestimated cause of electrical status epilepticus in sleep?

Objective: Christianson syndrome (CS) is a syndromic form of X-linked severe ID associated to progressive microcephaly, ataxia, autistic behaviour, near absent speech and epilepsy in 90% of cases. The natural history of seizures and EEG abnormalities is insufficiently known. We report two additional families including patients with electrical status epilepticus in sleep (ESES) previously described in only two patients. Methods: We describe two unrelated families with CS caused by SLC9A6 mutations (one frameshift and one partial deletion), each including two boys. One family has been followed for over 20 years, providing insights into the natural course of epilepsy in this syndrome. Results: Onset of epilepsy occurred within the first two years of life in all patients. Seizures were of various types. First EEG recordings showed abnormal fast-background activity with poor antero-posterior organization as reported in the literature. In the two boys with a 20 year follow-up, epilepsy was drug-resistant during childhood, then became less active in early adolescence. EEG background rhythm slowed over time to 4–5Hz after the age of 8 years. One patient from the first family fulfilled the electrical criteria of ESES, between 6 and 8 years of age, while sleep-EEG had not been performed in his brother during this range of age. ESES was diagnosed in the older brother from the second family since the age of 4 years and 10 months. The four boys presented severe ID, autistic features with agitation limiting frequent sleep EEG recordings. Conclusion: Our observations and previous reports, suggest that ESES might be an underestimated feature in CS and may participate to the psychomotor degradation of the patients. Interestingly, SLC9A6 mutations underlie dysfunction in the sodium-hydrogen exchanger NHE6 implicated in plasticity of glutamatergic synapses. Sleep EEGs should be performed in these infants between 4 and 8 years of age.

X-linked Christianson syndrome: heterozygous female Slc9a6 knockout mice develop mosaic neuropathological changes and related behavioral abnormalities.

ABSTRACTChristianson syndrome (CS) is an X-linked neurodevelopmental and neurological disorder characterized in males by core symptoms that include non-verbal status, intellectual disability, epilepsy, truncal ataxia, postnatal microcephaly and hyperkinesis. CS is caused by mutations in the SLC9A6 gene, which encodes a multipass transmembrane sodium (potassium)-hydrogen exchanger 6 (NHE6) protein, functional in early recycling endosomes. The extent and variability of the CS phenotype in female heterozygotes, who presumably express the wild-type and mutant SLC9A6 alleles mosaically as a result of X-chromosome inactivation (XCI), have not yet been systematically characterized. Slc9a6 knockout mice (Slc9a6 KO) were generated by insertion of the bacterial lacZ/β-galactosidase (β-Gal) reporter into exon 6 of the X-linked gene. Mutant Slc9a6 KO male mice have been shown to develop late endosomal/lysosomal dysfunction associated with glycolipid accumulation in selected neuronal populations and patterned degeneration of Purkinje cells (PCs). In heterozygous female Slc9a6 KO mice, β-Gal serves as a transcriptional/XCI reporter and thus facilitates testing of effects of mosaic expression of the mutant allele on penetrance of the abnormal phenotype. Using β-Gal, we demonstrated mosaic expression of the mutant Slc9a6 allele and mosaically distributed lysosomal glycolipid accumulation and PC pathology in the brains of heterozygous Slc9a6 KO female mice. At the behavioral level, we showed that heterozygous female mice suffer from visuospatial memory and motor coordination deficits similar to but less severe than those observed in X-chromosome hemizygous mutant males. Our studies in heterozygous Slc9a6 KO female mice provide important clues for understanding the likely phenotypic range of Christianson syndrome among females heterozygous for SLC9A6 mutations and might improve diagnostic practice and genetic counseling by helping to characterize this presumably underappreciated patient/carrier group.

Inaugural Christianson Syndrome Association conference: families meeting for the first time.

Christianson syndrome (CS) is an X-linked neurodevelopmental disorder caused by deleterious mutations in SLC9A6. Affected families organized the inaugural Christianson Syndrome Association conference to advance CS knowledge and develop questions that may be prioritized in future research.

X-linked Angelman-like syndrome caused by Slc9a6 knockout in mice exhibits evidence of endosomal-lysosomal dysfunction.

Mutations in solute carrier family 9 isoform 6 on chromosome Xq26.3 encoding sodium–hydrogen exchanger 6, a protein mainly expressed in early and recycling endosomes are known to cause a complex and slowly progressive degenerative human neurological disease. Three resulting phenotypes have so far been reported: an X-linked Angelman syndrome-like condition, Christianson syndrome and corticobasal degeneration with tau deposition, with each characterized by severe intellectual disability, epilepsy, autistic behaviour and ataxia. Hypothesizing that a sodium–hydrogen exchanger 6 deficiency would most likely disrupt the endosomal–lysosomal system of neurons, we examined Slc9a6 knockout mice with tissue staining and related techniques commonly used to study lysosomal storage disorders. As a result, we found that sodium–hydrogen exchanger 6 depletion leads to abnormal accumulation of GM2 ganglioside and unesterified cholesterol within late endosomes and lysosomes of neurons in selective brain regions, most notably the basolateral nuclei of the amygdala, the CA3 and CA4 regions and dentate gyrus of the hippocampus and some areas of cerebral cortex. In these select neuronal populations, histochemical staining for β-hexosaminidase activity, a lysosomal enzyme involved in the degradation of GM2 ganglioside, was undetectable. Neuroaxonal dystrophy similar to that observed in lysosomal disease was observed in the cerebellum and was accompanied by a marked and progressive loss of Purkinje cells, particularly in those lacking the expression of Zebrin II. On behavioural testing, Slc9a6 knockout mice displayed a discrete clinical phenotype attributable to motor hyperactivity and cerebellar dysfunction. Importantly, these findings show that sodium–hydrogen exchanger 6 loss of function in the Slc9a6-targeted mouse model leads to compromise of endosomal–lysosomal function similar to lysosomal disease and to conspicuous neuronal abnormalities in specific brain regions, which in concert could provide a unified explanation for the cellular and clinical phenotypes in humans with SLC9A6 mutations.

Christianson syndrome in a patient with an interstitial Xq26.3 deletion

Interstitial deletions of chromosome band Xq26.3 are rare. We report on a 2-year-old boy in whom array comparative genomic hybridization analysis revealed an interstitial 314 kb deletion in Xq26.3 affecting SLC9A6 and FHL1. Mutations in SLC9A6 are associated with Christianson syndrome (OMIM 300243), a syndromic form of X-linked mental retardation (XLMR) characterized by microcephaly, severe global developmental delay, ataxia and seizures. FHL1 mutations cause Emery-Dreifuss muscular dystrophy (OMIM 310300), X-linked myopathy with postural muscle atrophy (XMPMA, OMIM 300696), scapuloperoneal myopathy (OMIM 300695), or reducing body myopathy (OMIM 300717, 300718). The clinical problems of the patient reported here comprised severe intellectual disability, absent speech, ataxia, epilepsy, and gastroesophageal reflux, and could mostly be attributed to SLC9A6 insufficiency. In contrast to the majority of reported Christianson syndrome patients who were microcephalic, this patient was normocephalic, but his head circumference had decelerated from the 50th centile at birth to the 25th centile at the age of 2 ²/¹² years. Muscle problems due to the FHL1 deletion are not to be expected before late childhood, which is the earliest age of onset for FHL1 associated Emery-Dreifuss muscular dystrophy. This patient broadens the spectrum of SLC9A6 mutations and contributes to the clinical delineation of Christianson syndrome. This is also the first patient with a deletion affecting both SLC9A6 and the complete FHL1 gene.

A loss‐of‐function mutation in the SLC9A6 gene causes X‐linked mental retardation resembling Angelman syndrome

SLC9A6 mutations have been reported in families in whom X-linked mental retardation (XMR) mimics Angelman syndrome (AS). However, the relative importance of SLC9A6 mutations in patients with an AS-like phenotype or XMR has not been fully investigated. Here, the involvement of SLC9A6 mutations in 22 males initially suspected to have AS but found on genetic testing not to have AS (AS-like cohort), and 104 male patients with XMR (XMR cohort), was investigated. A novel SLC9A6 mutation (c.441delG, p.S147fs) was identified in one patient in the AS-like cohort, but no mutation was identified in XMR cohort, suggesting mutations in SLC9A6 are not a major cause of the AS-like phenotype or XMR. The patient with the SLC9A6 mutation showed the typical AS phenotype, further demonstrating the similarity between patients with AS and those with SLC9A6 mutations. To clarify the effect of the SLC9A6 mutation, we performed RT-PCR and Western blot analysis on lymphoblastoid cells from the patient. Expression of the mutated transcript was significantly reduced, but was restored by cycloheximide treatment, indicating the presence of nonsense mediated mRNA decay. Western blot analysis demonstrated absence of the normal NHE6 protein encoded for by SLC9A6. Taken together, these findings indicate a loss-of-function mutation in SLC9A6 caused the phenotype in our patient.

Natural history of Christianson syndrome

Christianson syndrome is an X-linked mental retardation syndrome characterized by microcephaly, impaired ocular movement, severe global developmental delay, hypotonia which progresses to spasticity, and early onset seizures of variable types. Gilfillan et al.2008] reported mutations in SLC9A6, the gene encoding the sodium/hydrogen exchanger NHE6, in the family first reported and in three others. They also noted the clinical similarities to Angelman syndrome and found cerebellar atrophy on MRI and elevated glutamate/glutamine in the basal ganglia on MRS. Here we report on nonsense mutations in two additional families. The natural history is detailed in childhood and adult life, the similarities to Angelman syndrome confirmed, and the MRI/MRS findings documented in three affected boys.

Dual degradation mechanisms ensure disposal of NHE6 mutant protein associated with neurological disease

Clinical features characterizing Angelman syndrome, previously shown to be caused by disruption of UBE3A, were recently also described in neurologically disabled patients with mutations in SLC9A6, which encodes the Na+/H+ exchanger NHE6. In the present work we have focused on NHE6Δ255–256, the protein product of a specific 6-bp patient deletion in SLC9A6. To resolve the molecular mechanism causing the cellular dysfunction associated with this mutant, we have characterized its intracellular behaviour in comparison to wild type NHE6. Our study demonstrates that NHE6Δ255–256 is much less stable than the wild type protein. Whereas wild type NHE6 is transported to the plasma membrane and early endosomes and remains stable, NHE6Δ255–256 is degraded via two independent pathways mediated by proteasomes and lysosomes, respectively. Depletion of NHE6 had no detectable effect on endosomal pH, but co-depletion of NHE6 and the closely related NHE9 caused enhanced acidification of early endosomes. Our results suggest that NHE6 participates in regulation of endosomal pH and provides a cellular basis for understanding the loss of NHE6 function leading to a neurological phenotype resembling Angelman syndrome.

SLC9A6 Mutations Cause X-Linked Mental Retardation, Microcephaly, Epilepsy, and Ataxia, a Phenotype Mimicking Angelman Syndrome

Linkage analysis and DNA sequencing in a family exhibiting an X-linked mental retardation (XLMR) syndrome, characterized by microcephaly, epilepsy, ataxia, and absent speech and resembling Angelman syndrome, identified a deletion in the SLC9A6 gene encoding the Na(+)/H(+) exchanger NHE6. Subsequently, other mutations were found in a male with mental retardation (MR) who had been investigated for Angelman syndrome and in two XLMR families with epilepsy and ataxia, including the family designated as having Christianson syndrome. Therefore, mutations in SLC9A6 cause X-linked mental retardation. Additionally, males with findings suggestive of unexplained Angelman syndrome should be considered as potential candidates for SLC9A6 mutations.