2.1 Ethical compliance and clinical examination

This study was approved by the Institutional Ethics Board of Jiangxi Provincial Children's Hospital (no. JXSETYY-YXKY-20210053), and informed consent was obtained from the patient's parents. Routine blood tests, tandem mass spectrometry, plasma ammonia, lactate β-Hydroxybutyric acid, electrolyte, blood glucose, electroencephalogram (EEGs), and magnetic resonance imaging (MRI) were performed, and all clinical information was collected and investigated. All the procedures were performed in accordance with the Declaration of Helsinki (2013 revision).

2.2 Genetic testing

Blood samples were collected from the patient and his family. Genomic DNAs was extracted from blood and purified by TIANamp Blood DNA Kit (DP348-02, TIANGEN, China) following the manufacturer's guide. After purification, 1ug genomic DNA from each sample was first sheered into fragments, purified, then captured using the XGen Exome Research Panel (IDT, USA) following the manufacturer's guide, finally the libraries were sequenced on the NovaSeq 6000 Sequencing Platform. The raw data generated by the sequencer were converted to to fastq format data by bcl2fastq (https://github.com/savytskanatalia/bcl2fastq). After removing low-quality reads and adaptors by TrimGalore (https://github.com/FelixKrueger/TrimGalore), the paired-end clean reads were mapped to the human reference genome (GRCh38/hg38) with Burrows-Wheeler Aligner (BWA) (Li & Durbin, 2009). Variant calling was performed using the Genome Analysis Toolkit (GATK) (Van der Auwera et al., 2013), and obtained variants were annotated using ANNOVAR (Wang et al., 2010). Variants located in exons or classical alternative splicing regions with a low frequency (<0.01) in public gnomAD database were obtained for further pathogenicity evaluation. The pathogenicity of the variants was classified according to the ACMG guidelines (Richards et al., 2015). Candidate pathogenic variants were confirmed by Sanger sequencing among the patient's family.

2.3 RNA extraction and MLC1 cloning procedures

Total RNA was extracted from blood using the RNAiso Plus kit (108/9109, TaKaRa) and then reverse-transcribed to cDNA for the construction of the expression vector as follows. Full-length open reading frame sequences of the wild-type and variant fragment of MLC1 were amplified from the cDNA from the proband and his father respectively by MLC1-GFP-BamHI-F (5′-GCTTGGTACCGAGCTCGGATCCATGACCCAGGAGCCATTCAGAGAG-3′) and MLC1-GFP-EcoRI-R (5′-GTGCTGGATATCTGCAGAATTCCTGGGCCATTTGCACCACGAC-3′). After validation by Sanger sequencing, the wild-type and variant fragments, pcDNA3.1-EGFP-P2A-MYC vector (constructed from the pcDNA3.1 (v790-20, addgene, MA, USA) as the plasmid backbone, reference sequence refers to the attachment) were digested with BamHI (1010S, Takara, Japan) and EcoRI (1040S, Takara, Japan) respectively following the manufacturer's guide. Then the wild-type and variant fragments were ligated to pcDNA3.1-EGFP-P2A-MYC vector respectively by T4 DNA ligase (2011A, Takara, Japan) following the manufacturer's guide. The vectors were transfected into the 293T cell line using Lipofectamine 2000 following the manufacturer's instructions. After 24 h, the cell was harvested for the following experiment.

2.4 qPCR

Total RNA was extracted from 293T cells using an RNA-easy Isolation Reagent kit (R071; Vazyme Biotech, China). First-strand cDNA was synthesized using (R212-01, Vazyme Biotech). qPCR analysis was performed using SYBR Green Real-Time PCR Master Mix (QPK-201, Toyobo, Osaka, Japan) in a CFX Connect Real-Time PCR System (Bio-Rad, Hercules, CA, USA). Data were obtained via a standard curve analysis and normalized to an internal control gene (ACTB).

2.5 Western blot

Proteins were extracted from 293T cells using the RIPA lysis and extraction buffer (P0013B, Beyotime, Shanghai, China). Protein levels were measured using a bicinchoninic acid (BCA) assay kit (P0010; Beyotime, Shanghai, China). Total protein (~20 μg/lane) was subjected to SDS-PAGE, before being transferred to a polyvinylidene difluoride membrane, blocked with 5% skim milk for 2 h, and incubated with mouse polyclonal anti-GFP tag (1:4000, AE012, Abclonal, Wuhan, China) and anti-GAPDH (1:1000, AF0006, beyotime, Shanghai, China) at 4°C overnight.

2.6 Statistical analyses

Data from independent experiments were presented as mean ± SEM. qPCR results were analyzed using a nonparametric test, and p < 0.05 were considered statistically significant. The significance between groups was calculated using GraphPad Prism 8 (GraphPad, USA).

3.1 Case presentation

A 4-year-and-11-month-old boy was admitted to our hospital for having experienced seizures following a fall. The seizures were characterized by confusion, both eyes fixed gaze deviation to left, diminished responsiveness, pale lips, rigid limbs, and limb twitching. The episode lasted approximately 10 min, and the patient exhibited a poor mental state and excessive sleepiness afterward.

At 1 year of age, he exhibited a large head circumference and started to walk around 2 years of age. He still had an unstable gait, quick walking with a tendency to lean forward, and poor jumping ability. His elder sister had a similar medical history, with a large head circumference (Figure 1a) and abnormal gait, diagnosed with “leukodystrophy.”

He exhibited slightly increased muscle tone, unsteady gait, Romberg sign, a positive finger-nose test indicating ataxia, and a hyperactive knee reflex. No rashes or depigmented patches were observed. Cardiovascular and abdominal examinations, routine biochemical markers, tandem mass spectrometry, plasma ammonia, lactate, β-hydroxybutyrate, electrolytes, and blood glucose showed no abnormalities.

Head MRI revealed the diffuse white matter swelling in the bilateral cerebral hemisphere accompanying frontal lobe cyst (Figure 1b(1,2)), temporal lobe cysts (Figure 1b(3)) and mild abnormal signals in the cerebellar white matter (Figure 1b(4)). Electroencephalography showed slow background activity. The developmental quotient (DQ) assessment indicated abnormalities (score: 44), with the most prominent impairment score (23.7) in gross motor skills.

Comprehensively considering the phenotype (head malformation, leukodystrophy, cerebellar ataxia, spasticity, epilepsy, etc.) and family history, he was highly suspected to have MLC. Blood samples were collected to perform exome sequencing for the identification of potential genetic variants.

3.2 Genetic testing

Trio whole-exome sequencing (WES) was performed to screen for potential pathogenic genetic factors associated with neurodevelopmental disorders and epilepsy. A homozygous variant of MLC1 [NM_015166.4: c.838_843delinsATTTTA (p.Ser280_Phe281delinsIleLeu)] was detected in both the proband and his older sister (Figure 1c). The variant was a 6 bp nucleotide deletion and another 6 bp nucleotide insertion, causing the substitution of the two SerPhe amino acids (280th and 281th nucleotides) to IleLeu. It was classified as variant of uncertain significance (VUS) for pathogenicity evidence: PM2_supporting (with a frequency of 0 in gnomAD), PM3_supporting (a homozygous variant detected in an autosomal recessive disorder), and PP1 (the variant was segregated in families). No other potentially pathogenic variants, including copy number variants (CNV), were detected.

3.3 Evaluation of the effects of the new variant on MLC1 expression

To evaluate the effect of this variant, wild-type and variant MLC1 overexpression experiments were designed. The cloning of wild-type and variant of MLC1 in the vector were first confirmed using Sanger sequencing (Figure 2a). No statistically significant differences of RNA levels were observed between the wild-type and variant by qPCR analysis (Figure 2b); however, a decreased level of the variant protein was observed when compared to the wild-type one by western blot experiment (Figure 2c).