A Novel Loss-of-function Pathogenic Missense Mutation in CASK Gene Causes Mental Retardation and Microcephaly with Pontine and Cerebellar Hypoplasia

Sixian Wu (  wusixian9911@163.com ) West China Second University Hospital, Sichuan University, https://orcid.org/0000-0001-9396-9635 Chuan Jiang West China Second University Hospital, Sichuan University, Jiaman Li Sichuan University West China School of Pharmacy Guohui Zhang West China Second University Hospital, Sichuan University, Ying Shen West China Second University Hospital, Sichuan University, Jing Wang West China Second University Hospital, Sichuan University,

Males affected by CASK mutations usually show more severe symptoms than females. These genetic issues are usually fatal in the womb for male embryos (7,14).
In the clinical screening for this study, researchers rst examined the case of an 11-month-old female patient with general developmental delay, microcephaly, and cerebellar hypoplasia. After screening by high-throughput sequencing detection technology of the whole exome, it was found that the patient had a novel missense mutation in CASK gene, at the location NM_003688.3: c.638T > G, p.L213R. In this study, researchers used bioinformatics methods in order to predict the pathogenicity and harm of this missense mutation in CASK. Researchers also examined the mRNA expression as well as protein expression of the mutation and predicted the structure of the protein. This study found that there is no difference between mRNA expression; however, amino acid mutations were found to cause downstream changes in the stability of the protein's spatial structure, which causes down-regulation of protein expression and loss of protein function.

Clinical summary
An unrelated natural couple brought an 11-month-old female with delayed development to the outpatient for genetic counseling ( Figure 1A). The proband exhibited features of general developmental delay, jaundice, clenched hands, and microcephaly. Her parents told us the patient had problems with sleeping, slow movement, and other developmental issues. Through testing, researchers found that the patient had increased muscle tone, dystonia, and cerebellar atrophy ( Figure 1B). Axial brain magnetic resonance imaging (MRI) was performed on the patient to obtain further information. The MRI revealed decreased size of both cerebellar hemispheres, widening of sulci in both cerebral hemispheres, and widening and deepening of cerebellar sulci ( Figure 1C). Researchers initially diagnosed the patient with mental retardation and microcephaly with pontine and cerebellar hypoplasia (MICPCH) depending on the speci c clinical characteristics. There were no phenotypic abnormalities observed in the parents of the patient.
According to the bioinformatics analysis, there was no previous report about this variation in most databases, including ExAC browser, 1000 genome project, or In-house Chinese-Control. The latest gnomAD database shows the frequency of this mutation as 0.000005520 (16) ( Table 1). In addition, this variant site is highly conserved in many species according to mutation taster (Figure 2A). PhastCons and PhyloP were used to evaluate the scores of amino acid sequence conservation. Scores indicated that this mutation site is highly conserved (Table 1). Moreover, this mutation was predicted to be pathogenic by the following bioinformatic tools: SIFT(17), PolyPhen-2(18), and M-CAP(19) ( Table 1). The above results found this mutation site to be pathogenic and well conserved.

Molecular genetic analysis
In order to further explore the molecular disease mechanism caused by the missense mutation of the CASK gene, wild-type and mutant plasmids were constructed and transferred into HEK-293T cells.
Researchers tested the mRNA expression ( Figure 3B) and protein expression ( Figure 3A&C) of wild and mutant type, and found that there was no signi cant difference in mRNA expression between the wild type and the mutant type while compared with the wild type, the protein expression of the mutant type is down-regulated.
Finally, researchers investigated the structural pattern of the protein after the amino acid arginine (R) was substituted for leucine (L) by PSIPRED (20)(21)(22). The results of protein structure prediction showed that the nuclear charge of the protein increased (ΔCharge=1) and the stability of the protein decreased (ΔΔG pred =1.857) after the mutation. At the same time, a random coil in the secondary structure is changed to β-sheet, which also affects its spatial structure ( Figure 2B). Decreased protein stability directly leads to protein degradation, which is also the reason for the down-regulation of protein expression. The above caused the loss of protein function.

Discussion
In this study, researchers examined the case of a female with a novel pathogenic missense mutation in CASK that is associated with MICPCH. Moreover, the signi cant results of decreased size of both cerebellar hemispheres, widening of sulci in both cerebral hemispheres, and widening and deepening of cerebellar sulci were observed on the results of MRI. The results of the WES sequencing show CASK gene had a missense mutation of CASK: NM_003688.3: exon7: c.638T>G: p.L213R. Although there was no signi cant difference found in the expression of the missense mutation in CASK by detecting the mRNA expression, the protein expression is down-regulation. By using the method of protein structure prediction, researchers found that when the leucine in the primary sequence of the protein was mutated to arginine, protein stability was reduced and secondary structure as well as spatial structure were changed. This resulted in reduced protein expression and loss of protein function.
All of the residues in the αF helix have been shown to be involved in a conserved spatial pattern. Kornev et al. (2008)(23) suggest that the residue at the position equivalent to 209 in CASK plays a critical role in the anchoring of the αH helix. This is purported to help stabilize the hydrophobic core around the αF helix. (12) Similarly, in this study, the leucine at position 213 in the amino acid sequence of the CASK protein is mutated to arginine. Since arginine is a basic amino acid and leucine is a non-polar amino acid (24), the amino acid change cause the change of maintenance of the third structure level in the protein (25). The reduction of hydrophobic force may not stabilize the hydrophobic core around the αF, which can greatly disrupt the function of CASK as a protein kinase.
Generally, the types of mutations that cause MICPCH are nonsense mutations, splice site mutations, shifting-frame mutations, and missense mutations. These types of mutations usually result in a decrease in CASK protein expression. The loss of CASK expression was associated with a more severe MICPCH phenotype and likely caused the reduced viability or in utero lethality. This research once again con rmed the loss of function causes severe general developmental delay, microcephaly, and cerebellar hypoplasia.
And it is rare that changed protein structure and increased instability in CASK protein leads to downregulation of protein expression and loss of function. These ndings extend the variety of genetic alterations causing CASK mutations. Moreover, this research is of great signi cance for understanding the pathogenicity of CASK point mutation. This study further reveals the key role of CASK in MICPCH development, and helps provide genetic explanations for the etiology of CASK mutation in MICPCH affected patients.

Conclusions
In conclusion, this research rst reports that the novel pathogenic missense mutation in CASK (NM_003688.3: exon7: c.638T > G: p.L213R) in the 11-month-old female caused mental retardation and microcephaly with pontine and cerebellar hypoplasia (MICPCH). This study also determined the bioinformatic content of this mutation site and explored there was no differences in mRNA expression while protein expression was down-regulation. Finally, the researchers analyzed the structural changes of the protein and con rmed that the cause of the disease was the down-regulation of the protein caused by amino acid mutations that changed the stability of the protein's structure.

Subjects
Peripheral blood samples were obtained from the proband and her family after informed consent was signed by the patient and all family members. This experiment on human subjects was approved by the Ethical Review Board of West China Second University Hospital, Sichuan University. The patient's brain MRI results were evaluated by the pediatric neuroradiologist.
Whole-exome sequencing Genomic DNA was extracted from peripheral blood leukocytes using a whole blood DNA puri cation kit (TIANGENE). For WES, exons were captured from 1 μg genomic DNA using high-throughput sequencing detection technology for the whole exome provided by the manufacturer. The Verita Trekker® variant site detection system and Enliven® variant site annotation interpretation system independently developed by Berry Genomics were used to analyze the data. Functional annotation was performed through ANNOVAR, and data were ltered by public databases such as ExAC, 1000 Genomes Project, and GnomAD . were placed on a shaker at 37°C overnight. After shaking was complete, researchers extracted the plasmid from E. coli.
The HEK-293T cell line was obtained from the American type culture collection (ATCC, USA). The 293T cells were cultured in 6-well cell culture plates and 100 mm cell culture dishes (WHB, China) with basic DMEM medium containing 10% fetal bovine serum (Gibco, USA) and 0.1% penicillin/streptomycin in a humidi ed incubator at 37°C with 5% CO 2 . According to the experimental scheme, CASK plasmids were transfected into 293T cells for 24-48 h.

Quantitative PCR
The total RNA of the cell was extracted using TRIzol reagent (Invitrogen) and was converted to cDNA using a Revert Aid First-Strand cDNA Synthesis Kit (ThermoFisher). Quantitative PCR was performed using SYBR Premix Ex Taq II (TaKaRa) on an iCycler RT-PCR Detection System (Bio-Rad Laboratories).
The ΔΔCT method was used for data analysis. Each assay was performed in triplicate for each sample.
The GADPH gene was used as an internal control.
The primers of human GADPH are as follows: The primers of human CASK are as follows: 5′ GACTTGTAGCTGGAGGACGTG 3′ 5′ GATATGGCTCCACTGCCTTGG 3′

Western blotting
The proteins were extracted using radioimmunoprecipitation assay (RIPA) buffer that contained a protease and phosphatase inhibitor cocktail (Roche). Twenty microgram of the denatured proteins were separated with the use of 10% SDS-polyacrylamide gels and transferred to a polyvinylidene di uoride (PVDF) membrane (Millipore) for immunoblotting analysis. After blocking with Tris-buffered saline/Tween-20 (TBST) containing 5% bovine serum albumin (BSA) for 1 h at room temperature, the membranes were then incubated with the corresponding primary antibodies 1:50 anti-FLAG (HPA052219; Sigma-Aldrich) and 1:2000 anti-β-actin (T7451; Sigma-Aldrich) at 4°C. Samples were incubated overnight. The binding of the primary antibodies was visualized using horseradish peroxidase-conjugated goat antirabbit or antimouse IgG (1:10,000, ZSGB-BIO, China). The signal intensities were measured using ECL (1305702; Millipore Corporation, Billerica, USA) and image analysis software (ImageJ, NIH).

Immuno uorescence
The cells transferred into the plasmid were cultured in a glass-bottom dish. Then the cell samples were xed onto slides with using 4% paraformaldehyde for 10 min, the followed by slides were washed by with PBS. The slides were permeabilisedzed with 0.3% Triton X-100 and blocked with 5% BSA in PBS. Next, the slides were then incubated with the corresponding primary antibodies (anti-FLAG 1:50) overnight at 4 °C. Second on the following day, the slides were washed by PBS, then incubated with Alexa Fluor 488 (1:500) (A21206; Thermo Fisher) labelled secondary antibodies for 1h at room temperature, and then counterstained with 4,6-diamidino-2-phenylindole (DAPI,) (Sigma-Aldrich) to label the nuclei. Images were obtained using a laser scanning confocal microscope, 60X (Olympus).