In 2008, a study identified four mutations in SLC9A6 in different families that altered the function of NHE6. A total of three fragment deletions of the X chromosome and 47 pathogenic mutations were identified, of which 13 were intron splice site variants; de novo chromosomes or mutations accounted for 44.00% (22/50; Figure 3C). Lizarraga et al. demonstrated that pathogenic mutations lead to loss of protein function through various mechanisms:most mutations led to loss of mRNA due to nonsense-mediated mRNA decay. The exon mutation profile shows that exon 12 is a hot spot for pathogenic mutations and harbors 43.8% of all variants. A 3-year-old Chinese patient with CS had a splicing mutation (c.1463-1G > A) in SLC9A6, which was verified by in vitro transcription. This intron 11 mutation caused exon 12 skipping. The patient presented with seizures, intellectual disability, microcephaly, feeding difficulties, hyperkinesia, ataxia, and cerebellar atrophy. In this report, the mutation identified is a classical splice site mutation in intron 11. Plasmids with a splice variant (c.1366 + 1G > C) of SLC9A6 was constructed using a minigene splicing assay to transcribe the two mRNA products. The main splicing forms included a partial deletion of 42 bp in exon 11, which may lead to the formation of a truncated protein. A minor splicing form includes a partial deletion of 12 bp in exon 10 and exon 11 skipping, which may lead to an amino acid shift to form a truncated protein. This study speculates that this mutation results in the aberrant splicing of SLC9A6, forming truncated proteins and contributing to CS development. However, the main clinical phenotypes of the patient reported in this study were seizures and developmental regression, which were milder than those reported for the 3-year-old Chinese patient with CS, and no genotype-phenotype correlations were observed in this study.
The NHE protein family has nine members. NHE1-5 are present in the cytoplasmic membrane, whereas NHE6-9 are present in organelle membranes. NHE proteins have an N-terminal domain of 10-12 transmembrane helices and a hydrophilic C-terminal domain, which is regulatory. NHE6, a dodecameric transmembrane protein, is localized in early recycling endosomes, but not in lysosomes, and transiently appears on the plasma membrane. Overexpression of NHE isoforms increases the luminal pH of the compartment in which the NHE is located; NHE6 changes the early endosomal membrane endoluminal pH from slightly acidic to neutral. Thus, a possible consequence of NHE6 inactivation is a decrease in pH of the endosomal recycling compartment. NHE6 overexpression reduces monovalent ion content, which may affect protein folding and transport. Neurons with SLC9A6 knockout had fewer lysosomes in the cytoplasm and reduced activity of lysosomal proteases; moreover, autophagic flow was reduced by >50% in SLC9A6 knockout neurons compared with wild-type neurons. Membrane trafficking from recycling endosomes is essential for the growth and maintenance of dendritic spines in long-term potentiation, which is thought to be the molecular basis of learning and memory. Thus, mutations in SLC9A6 likely affect long-term potentiation. The female SLC9A6 mutation carrier phenotypic spectrum encompasses learning difficulties, speech and language delay, mild-to-moderate intellectual disability, and behavioral issues. Consistent with many X-linked disorders, skewed X-inactivation may play a role in the clinical presentation of SLC9A6 mutations in female carriers. Sikora et al. created a heterozygous SLC9A6 knockout female mouse that expressed the mutant SLC9A6 allele to follow the same neuroanatomic distribution and cell-specific patterns as hemizygous mutant males. The heterozygous knockout female mice developed progressive behavioral and neuropathological abnormalities that were similar to but milder than hemizygous male knockout mice.
Nearly 98.86% of patients with CS have epilepsy. Their EEG background rhythms were normal or abnormal with fast waves, and interictal epileptiform discharges were focal, multifocal, or widespread. Mathieu et al. found that electrical status epilepticus during sleep is a constituent feature of patients with CS, especially in the case of intellectual disability, autism, and severe speech delay, which may contribute to the diagnosis of CS. A detailed analysis of the clinical data of 53 patients with epilepsy-related conditions revealed that seizures started earlier than 3 years of age, with tonic-clonic episodes in nearly all cases. Other common seizure types included atypical absence, tonic seizures, myoclonic seizures, and focal onset seizures. Some patients were diagnosed with Lennox-Gastaut syndrome and West syndrome (Supplementary Table 4). These patients had poor outcomes despite treatment with multiple antiseizure medications, although VPA, LEV, clonazepam, and lamotrigine were more effective and more frequently used. Developmental regression after onset of seizure clusters was reported in some cases. Ikeda et al. reported a case of developmental regression at the age of 3 years following resistance to antiseizure medication. We speculate that this resulted from progressive brain dysfunction due to uncontrollable and frequent seizures. The patient in this study had an acute onset of seizures at the age of 1 year and 3 months; the seizures had a fluctuating course and occurred in clustered outbreaks. The background rhythm of VEEG in this case was normal with multifocal spike and wave complexes and sharp waves, which was consistent with the reported cases. Before the onset of CS, the patient could stand alone, walk with assistance, and pinch objects with the thumb and index finger and had recognition performance. After the onset of CS, the patient exhibited developmental regression and could not sit up, crawl, or stand unaided. He received treatment with phenobarbital, diazepam, and OXC, which did not improve his condition.
Nearly 35.09% of patients with CS showed cerebellar atrophy on MRI. The hemisphere and cerebellar vermis were the most frequently affected areas. Bilateral temporal and occipital subarachnoid widening have been reported in several cases. Neuropathological studies have shown that cerebellar atrophy was caused by neuronal loss of Purkinje cells, and extensive progressive loss of Purkinje cells and gelatinization were observed in the cerebellum of SLC9A6 mutant mice. For the patient in this study, a brain MRI performed at the age of 1 year and 2 months showed a prominent cisterna magna. The patient will be regularly followed-up with MRI. Additionally, the patient had PVCs and ventricular parasystole; however, arrhythmias have not been reported in patients with CS. We hypothesize that the arrhythmia could be related to the myocarditis lesions in the apical and basal segments of the left ventricle, and further investigation is required.
There are no effective treatments for CS. Antiseizure medications can be administered to patients with CS who have epileptic seizures according to clinical manifestations, and adjusted based on the therapeutic effect. Medically intractable epilepsy can be managed with a ketogenic diet, and rehabilitation training is available for patients with psychomotor retardation. There are two future directions for CS treatment: gene therapy and treatment according to CS pathogenesis. Ouyang et al. demonstrated that loss of NHE6 results in the over-acidification of the endosomal compartment and attenuates tropomyosin receptor kinase B (TrkB) signaling; mouse brains with disrupted NHE6 displayed reduced axonal and dendritic branching, synapse number, and circuit strength. Functional defects associated with TrkB signal attenuation due to NHE6 deficiency can be rescued by high levels of exogenous TrkB. The reported cases indicate a link between phenotype and clinical outcome in CS; patients with developmental regression, frequent uncontrollable seizures, and complicated abnormalities of other systems, such as the digestive and respiratory systems, have limited life expectancy. Further studies must verify this association and clarify its mechanistic basis.