The patient received medical care at Shenzhen Children’s Hospital, where various clinical diagnostic examinations, including MRI and EEG, were conducted. Medications prescribed for the patient included levetiracetam, sodium valproate, zonisamide, and nitrazepam. Additionally, the patient was assessed using the Wechsler Intelligence Scale, received ketogenic diet therapy, and underwent adjunctive therapy with a skin patch. Furthermore, trio whole-exome sequencing tests were performed.

Genomic DNA was extracted from peripheral blood samples. Trio whole-exome sequencing (Trio-WES) was performed using a SureSelect Human All Exon kit V6 (Agilent Technologies, Santa Clara, CA) on the Illumina Novaseq6000 at an average depth of 100× at Berry Genomics, Beijing, China.

RNA extraction was carried out using TRIzol reagent, ensuring the retrieval of total RNA. To enrich the mRNA, a subsequent step was performed, followed by purification. The purified mRNA was then fragmented, and reverse transcription was conducted to synthesize complementary DNA (cDNA). The generated cDNA underwent end repair and 3′-adenylation processes. Adapter sequences were ligated to the cDNA molecules, facilitating their amplification and purification through PCR. The PCR products were then converted into single-stranded DNA, which were further transformed into closed-loop structures. Subsequently, a sequencing library was prepared, and finally, sequencing was performed.

Total RNA was extracted from peripheral blood using the Blood (Liquid Sample) Total RNA Rapid Extraction Kit (RP4001, Bioteke); this was followed by genome digestion and cDNA reverse transcription using the HifairTM 1st Strand cDNA Synthesis SuperMix (11123ES70, YEASEN) kit. The system was heated to 95°C for 5 min, followed by PCR amplification using PrimerSTAR MAX DNA Polymerase (R045A, TaKaRa) at 95°C for 30 s, 57°C for 30 s, and 72°C for 90 s (35 cycles), and then 72°C for 5 min. Finally, Sanger sequencing was performed to verify the results.

To analyze how the identified variants affected splicing, minigene assay was carried out; the specific steps were as follows. First, minigene trapping and digestion site introduction were performed. Second, the recombinant vectors were transiently transfected into HeLa and 293T cell lines; the transfection step was performed according to the liposome instructions (Rapid Plasmid Mini Kit, 1005250, SIMGEN) and the samples were collected after 48 h (DNA Gel Extraction Kit, 2001250, SIMGEN). Next, total RNA was extracted from cell samples using an RNA extraction kit (Trizol RNAiso PLUS, 9109, TaKaRa, Kusatsu, Japan). After determination of the concentration, cDNA was synthesized by reverse transcription with equal amounts of RNA. Finally, PCR amplification was performed using the flanking primers (pcDNA3.1-F and pcDNA3.1-R) on the minigene vector, and the resulting amplified gene transcript bands were detected via agarose gel electrophoresis. Each band was then recovered separately for Sanger sequencing.

The patient was a 12-year-old girl and the first child to non-consanguineous, healthy parents, born normally. Her birth weight was 3150 g. She began to walk independently at 14 months of age. No obvious abnormalities were observed before the age of two. At 2 years old, rapid shaking and tremor of hands when holding objects were observed. The symptoms were aggravated during mood fluctuations and were dramatically improved upon administration of nitrazepam.

When the patient was 6 years old, she developed a frequent rapid hand tremor; therefore, she underwent monitoring via brain magnetic resonance imaging (MRI) and electroencephalography (EEG) at a local hospital. Brain MRI was normal, but EEG revealed interictal generalized spike-and-wave patterns and myoclonic seizures. The patient was treated with levetiracetam and sodium valproate, which resulted in improvement in symptoms. In addition, moderate myopia was noticed at the time.

At 10 years old, the patient presented with frequent blinking, accompanied with slightly worsened cognition and school performance, with a Wechsler intelligence score of 77. She could walk and speak normally. Although she still had slight tremor during fine hand movements, she could write in Chinese that was easily recognizable. EEG monitoring revealed tonic seizures: in terms of semiology, these presented as frequent blinking in clusters along with low-amplitude, generalized fast activities in ictal EEG (Figure 1). Repeated brain MRI was normal. The patient was switched to a ketogenic diet, which was followed by improvement in cognition and a slight reduction in blinking. Trio whole-exome sequencing and mitochondrial gene sequencing were carried out. She also received perampanel add-on therapy, resulting in a 60% reduction in blinking frequency and slight improvement in limb tremor. Zonisamide was given to aid in further control of tonic seizures, which produced a limited reduction in seizure frequency. At the age of 12, her height was 152 cm (−0.2 SD) and her weight was 32 kg (−1.8 SD). She continued to exhibit blinking and hand tremor and was taking perampanel, zonisamide, and nitrazepam.

In order to further investigate the potential genetic causes of the disorder, family whole-exome sequencing was performed on the proband and her parents. SNV and InDel results showed that there were two heterozygous variants of NUS1, namely, c.791 + 6T>G and c.537T>A. The missense variant c.537T>A was present in both the patient and her healthy father, but was absent in her sister and mother (Supplementary Figure S1), suggesting that it was not likely to be the cause of the disease. The other intronic variant, c.791 + 6T>G, was identified exclusively in the patient and was not found in the mother, father, or sister (Figure 2), supporting a co-segregation of the variant with disease. Additionally, this intronic variant was not found in the Shenzhou Genome database, the human Exon Database (ExAC), the reference population of the 1000 Genomes Project (1000G), or the Population Genome Mutation Frequency Database (gnomAD), suggesting that it was a novel variant. Therefore, we considered the variant c.791 + 6T>G to be the likely cause of the disease.

In order to verify the pathogenicity of NM_138,459.5: c.791 + 6T>G, in vitro and in vivo experiments were conducted to identify its functional consequences. Samples from the patient’s healthy parents were used as controls. RNA-Seq results showed that the patient (II-1) showed both inclusion and exclusion of Exon 4, whereas the healthy controls (I-1 and I-2) exhibited only normal inclusion of Exon 4 (Figure 3A). In vivo RT-PCR and Sanger sequencing confirmed that both the father and the mother had a single normal band ‘a’ (expected size 477bp), but the patient had two bands; band ‘a’ was the same size as expected, and band ‘b’ was smaller than band ‘a’ due to skipping of Exon 4 (Figure 3B). Then two in vitro expression constructs of Exon 4 (pcMINI-C and pcMINI) and the flanking sequences that contained the variant site were assembled and minigene splicing assays were carried out in 293T and HeLa cells. In the wild type constructs, there were bands consistent with the expected size for Exon 4 inclusion, while in the variant constructs, bands 100 bp smaller were observed due to skipping of Exon 4 (Figure 3C). Taken together, these experimental results suggest that the c.791 + 6T>G variant in NUS1 does affect the normal splicing of mRNA, causing Exon 4 to be skipped.

The c.791 + 6T>G variant of NUS1 gene is a new and rare splicing variant, and its detrimental impact on splicing has been demonstrated through in vitro and in vivo functional experiments. According to the pathogenicity classification guidelines of the American College of Medical Genetics and Genomics (ACMG), the c.791 + 6T>G variant is classified as a pathogenic variant. The pathogenicity rating (PS2+PM2+PS3 Moderate = P) was established as follows.

1) PS2 requires confirmation of the new variant through parental verification and absence of family history. The results of family exome sequencing confirmed that the c.791 + 6T>G variant is a de novo variant in this case.