Christianson syndrome: A novel splicing variant of SLC9A6 causes exon skipping in a Chinese boy and a literature review

Abstract Background Variants in the endosomal solute carrier family 9 member A6 (SLC9A6)/(Na+,K+)/H+ exchanger 6 (NHE6) gene have been linked to epilepsy, speech loss, truncal ataxia, hyperkinesia, and postnatal microcephaly. Methods In the present study, we evaluated genetic alterations in a 3‐year‐old Chinese boy displayed features of epilepsy, psychomotor retardation, microcephaly, low body weight, difficulty in feeding, excessive movement, attention loss, ataxia, and cerebellar atrophy and his healthy family using WES method. The identified variant was further confirmed by Sanger sequencing method. Finally, minigene assays were used to verify whether the novel SLC9A6 intronic variant influenced the normal splicing of mRNA. Results We identified a novel hemizygous splicing variant [NM_001042537.1: c.1463‐1G>A] in SLC9A6 by trio‐based exome sequencing. The minigene expression in vitro confirmed the splicing variant altered a consensus splice acceptor site of SLC9A6 intron 11, resulting in skipping over exon 12. Conclusions Our finding extends the catalog of pathogenic intronic variants affecting SLC9A6 pre‐mRNA splicing and provides a basis for the genetic diagnosis of CS.

microcephaly. 1,5,6 Furthermore, these core phenotypic features can also be accompanied by secondary symptoms, such as autistic behaviors, poor weight gain, feeding difficulties, motor regression, sleep disturbances, squint, high pain threshold, hypotonia, gastroesophageal reflux, and different degrees of cerebellar atrophy (CA). [7][8][9] Contrast to male, female variant carriers show milder phenotypes with variable penetrance. 4,10 Affected CS patients show a remarkable neurological symptom may be due to high-level expression of mutant SLC9A6 gene in the central nervous system. 11,12 To date, few Chinese CS patients have been reported. Here, we report a Chinese boy presenting with Christianson syndrome due to novel splicing variant NM_001042537.1: c.1463-1G>A in the SLC9A6 gene. The variant is not mentioned in ClinVar, dbSNP, 1000genomes, NCBI, ExAC, or HGMD database. We performed minigene splicing assay to validate the functional effect of variant in SLC9A6 gene to determine whether the hemizygous site is a pathogenic site for the affected boy.

| Patient and ethics
The present study was approved by the Ethics Committee of Northwest women's and children's hospital (Xi'an, China). Written informed consent was obtained from the parents.

| Samples and DNA extraction
Genomic DNA was extracted from 2-3 ml EDTA peripheral blood sample using BloodGen Midi Kit (CWBIO), according to the manufacturer's protocol.

| Variant screening
Library enrichment for whole exome sequencing was conducted using SeqCap EZ Exome v3 kit (Roche NimbleGen). Then, the enriched samples were sequenced by using an Illumina Hiseq 2500 (Illumina). The variants which is presented at least 20% of all reads were identified as quality variants. Thus, all quality variants were evaluated by bioinformatic tools including Polyphen2, PROVEAN, and MaxEntScan. The pathogenicity of all quality variants was accessed by The American College of Medical Genetics and Genomics (ACMG) Standards and Guidelines. 13 The pathogenic variant was validated by Sanger sequencing.

| Transcription analysis
SLC9A6 fragments containing intron11, exon12, and intron12 were amplified from DNA of patient and control by nested PCR. The first PCR was performed using genomic DNA as a template, with 37527-SLC9A6-F and 39956-SLC9A6-R as primers; the second PCR was performed using products from the first PCR as a template, with 37833-SLC9A6-F and 39651-SLC9A6-R as primers; the third PCR was performed using PCR products from the second round as a template, with pcMINI-SLC9A6-KpnI-F and pcMINI-SLC9A6-BamHI-R as primers. The primer sequences were listed as Table 1.
Restriction enzymes Kpn I and BamH I were used to digest amplified products, and the digestion products were ligated to pcMINI plasmid to obtain the pcMINI-SLC9A6-wt and pcMINI-SLC9A6-mut plasmid ( Figure 4A). The recombinant plasmids were then transfected into human embryonic kidney cells (HEK-293T) and human cervical cancer cells (HeLa) by lipofectamine 2000 (Invitrogen). After 48 h, cells were harvested and RNA was extracted by RNAiso Plus (TAKARA). cDNA was synthesized using PrimeScript RT Master Mix (TAKARA). Finally, the cDNA was identified using 1.8% agarose gel electrophoresis and verified through sequencing.

| Clinical case report
The boy was born in full-term normal delivery without anoxia and asphyxia history. His birth weight, length, and occipitofrontal circumference (OFC) were reported to be within normal range. There were no family histories of mental illness, genetic illness, epilepsy, and febrile seizures. His six-month-old sister was in good health. His parents were both Han Chinese.
He started a seizure at the age of 12 months, characterized by general tonic-clonic seizure, and has been treated with topiramate (TPM) firstly. TPM was stopped using without permission from doctor after half a month. However, 3 months later, he had frequent general tonic-clonic seizures in clusters for 6 months, despite antiepileptic treatment by sodium valprorate (VPA). General tonic-clonic seizures were suddenly completely controlled by additional use of levoethylacetam (LEV) and repetitive transcranial magnetic stimulation (rTMS), with a seizure-free interval between 22 months old and 34 months old. During the absence of seizures, rTMS has been added only a few times and then discontinued, LEV was replaced with lamotrigine (LTG) because of his hyperkinesis and bad appetite after using 6 months, and LTG was subsequently replaced with Furthermore, EEG and MRI were rechecked. Awake EEG revealed that on the basis of diffuse irregular medium to high amplitude 4-6 Hz θ rhythm, middle to very high amplitude spikes, multiple spikes, spikes and slow waves complex, and rhythms were exploded frequently and asynchronously. The leads of frontal, occipital, and temporal were prominent. The right hemisphere was more obvious than the left. During sleep, epileptiform discharge was significantly increased than awake (see Figure 1). The brain MRI revealed hypoplasia of inferior parts of cerebellar vermis, and enlargement of inferior orifice of the fourth ventricle, and abnormal signals in the left frontal cortex. The other intracranial structures including brainstem, basal ganglia, and supratentorial brain structures were within normal limits ( Figure 2).

TA B L E 1 Primers for transcription analysis
Since he came out of our hospital, there has been a seizure-free interval of 5 months. During this period, his ambulation and appetite were a little better than before, but his language development and weight gain still changed little. He still had no autistic behavior, but he was easily irritated. He was more likely to suffer from upper respiratory infections than normal children, sometimes even had fever, but there was no seizure. General tonic-clonic, myoclonic, and atonic seizures were no longer appeared. But sleep seizures occurred occasionally again after 5 months, characterized by consciousness loss and upward gaze, without other abnormal movements, and relieved after about 3 minutes.
The dose of LEV was adjusted from 30 to 60 mg/kg every day. After each dose adjustment, the frequency of seizures would be significantly reduced, but sleep seizures were not controlled completely.

| The whole exome sequencing reveals a novel SLC9A6 splicing variant
Whole exome sequencing was performed on the proband and his parents and sister, and variant filtering was performed as described in the methods. Annotated whole exome sequencing data were ex- In vitro transcription analysis was performed to validate how the variant affect splicing products. Figure 4B showed that mutant plasmid encoded a shorter transcriptional products compared to the WT plasmid. The results of Sanger sequencing demonstrated the intron mutant lead to a complete skipping of exon 12 ( Figure 4C, Figure 4D).

F I G U R E 2 EEG findings. (A) Awake
EEG revealing on the basis of diffuse irregular medium to high amplitude 4-6 Hzθ rhythm, middle to very high amplitude spikes, multiple spikes, spikes and slow waves complex, and rhythms were exploded frequently and asynchronously. The leads of frontal, occipital, and temporal were prominent. The right hemisphere was more obvious than the left. (B) Sleep EEG revealing frequent unsynchronized paroxysm of middle to very high amplitude spikes, mutiple spikes, spikes and slow waves complex, and rhythms, over bilateral anterior-middle frontal, occipital, and temporal. The right hemisphere was more obvious. Epileptiform discharge was significantly increased than being awake

TA B L E 2 (Continued)
According to the previous reports, CS specific brain MRI presented as CA. CA occurred mainly in the inferior parts of vermis and cerebellar hemisphere, especially in the cerebellar vermis. 7 This patient has received two brain MRI scans at the age of 9 month and 3 year, respectively. The first brain MRI showed normal intracranial structures, but the later brain MRI showed the inferior parts of cerebellar vermis dysplasia and the inferior orifice of the fourth ventricle enlargement (Figure 1), which indicated that the cerebellum was slowly atrophy and its further development should be monitored during follow-up. It was reported that the occurrence of CA was mainly related to the extensive progressive loss and degeneration of cerebellar Purkinje cells caused by SLC9A6 gene variant in the CS. 21 The above results indicated that the occurrence of CA was progressive in the CS. According to the neuroimaging data of 17 CS patients, the average age of onset of CA was about 11 years old. 7 Therefore, although the brain MRI scans indicate no abnormality, the possibility of CS should also be considered in the patients with mental retardation less than 1 year of age. Their brain MRI should be monitored constantly and regularly after onset.
There are still some limitation in this study. On the one hand, transcription analysis in the patient was not investigated. It was reported that peripheral blood mRNA analysis is helpful for ascertainment of alternative splicing of SLC9A6 induced CS. 29 On the other side, the correlation between genotype and phenotype is still need further investigation.
In conclusion, few Chinese CS patients have been re-

ACK N OWLED G M ENTS
The authors thank the patient's parents for agreeing to this report and providing a lot of valuable information. This work was supported by National Natural Science Foundation of China (No. 81800787).

CO N FLI C T O F I NTE R E S T
The authors declare that they have no competing interests.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data used to support the findings of this study are included within the article. And the raw data used to support the findings of this study are available from the corresponding author upon request.