The proband, accompanied by his parents and siblings, was admitted to our Neurology Department at the First Affiliated Hospital of Fujian Medical University with the chief complaint of recurrent epileptiform seizure. Brain magnetic resonance imaging (MRI) scan, electroencephalogram (EEG), electrocardiogram (ECG), ambulatory blood pressure monitoring, and echocardiography were utilized to assist in the diagnosis of the disease. Diagnosis was confirmed by two experienced neurologists. Peripheral blood samples were collected from all family members, and genomic DNA was extracted using the QIAamp DNA Blood Mini Kit (QIAGEN, CA, USA) according to the manufacturer’s instructions. This study was approved by the ethics committee for Medical Research of the First Affiliated Hospital of Fujian Medical University (FYYY2006-01-1901), and written informed consent was obtained from all participants.

Whole-exome sequencing (WES) was performed on the proband using Agilent SureSelect V6 capture kits (Aglient Technologies Co., Ltd) on the Illumina HiSeq 2,500 platform. Raw data were converted to FASTQ files, and low-quality reads were eliminated. Clean FASTQ formatted sequences were aligned to the human reference genome using BWA-MEM. Variant calling was conducted using GATK haplotype caller, and annotation was performed using ANNOVAR. Only variants fufilling a recessive inheritance pattern with a frequency of less than 1% in the gnomAD population, combined with the preliminary clinical diagnosis, were screened.

To confirm the variants identified through a WES analysis, specific primers were designed for amplification of the WDR62 mutations. The targeted regions were polymerase chain reaction (PCR) amplified in a C1000 Touch Thermal Cycler (Bio-Rad). Following purification, the amplified products were electrophoresed on an ABI 3730xl automated DNA analyzer (PE Applied Biosystems, Foster City, CA) according to standard protocols. The sequencing results were aligned with reference genomes downloaded from Ensembl.¹

RNA extraction from blood samples of the proband and the negative control was performed using TRIzol reagent (Invitrogen, CA, USA) following the manufacturer’s recommendations. For cDNA synthesis, we used 1 μg of each RNA sample per 20 μL reverse transcription reaction using a R333 HiScript® III All-in-one RT SuperMix (Vazyme Biotech Co, Ltd). The forward primer 5’-CTGAGACTGACCCTGTCAAGTGCCT-3′ and the reverse primer 5’-ACAGGAAGGTGGAGACCAGCTCAGT-3′ were designed to amplify the target region. The products were then used for agarose gel electrophoresis imaging to analyze the splicing alterations of the pre-mRNA affected by the splicing site variation.

The pCAG-Flag plasmid was linearized with BamHI and XhoI restriction endonuclease (Thermo Fisher Scientific), and primers were designed to amplify the target fragment. Plasmids were constructed using the NEBuilder HiFi DNA Assembly Master Mix (New England Biolabs) according to the standard manufacturer’s protocols. Two plasmids were constructed: One contained Flag-tagged WDR62 cDNA with wild-type intron 30, and the other contained Flag-tagged WDR62 cDNA with intron 30 containing the c.4154-6C > G variant. HEK293T cells were transfected with 2 μg of plasmids using Lipofectamine 3,000 (Thermo Fisher Scientific). After 48 h, the transfected cells were harvested. Protein extraction and Western blotting analysis were conducted using standard protocols, and four biological replicates were collected for statistical analysis (t-test). Differences were considered statistically significant when p-values were less than 0.05 (*), 0.01 (**), 0.001 (***), or 0.0001 (****). “ns” indicates not significant.

The proband, a 25-year-old male from a family of consanguineous marriage, exhibited phenotypical and behavioral characteristics of primary microcephaly, recurrent epilepsy, intellectual disability, limping gait, single-word speech, and an inability to walk in a straight line. The pedigree of the family is shown in Figure 1A. At the age of 1 month, he experienced hyperpyrexia lasting for 1 week. Since the age of 5 months, he had recurrent seizures occurring approximately once a month, which could be triggered by stimuli such as sound, temperature changes, falling, and limb touch. At 1 year of age, radiological imaging revealed dysplasia of the left femoral head. The patient was unable to crawl during infancy and began sitting independently at 3 years and moved his body with support from a bench. He achieved independent walking at 6 years of age and learned to use chopsticks at 8 years of age. Due to delayed psychomotor development, he did not receive formal education and never attended school. Seven years ago, he was admitted to a local hospital and started antiepileptic therapy with valproate acid and lamotrigine, which partially controlled his symptoms, although intermittent attacks persisted. At the age of 25 years, clinical evaluation revealed the patient’s stature was short (154 cm), with measurements indicating left upper limb length of 71.3 cm, right upper limb length of 76 cm, left lower limb length of 81 cm, right lower limb length of 84 cm, and head circumference of 50.5 cm (Figure 1B).

MRI scans were performed on the proband to evaluate brain structures, revealing an obvious microcephaly with widened sulci and reduced gyri, thinning of the corpus callosum, particularly in the splenium, dysplasia of the right cerebral hemisphere with bilateral ventricles of unequal size, diffusely thickened cortex, loss of gray–white junction, and partial pachygyria. Schizencephaly was observed in the left parietal lobe, along with asymmetric atrophy of the mesencephalon, pontomedullary, and oblongata and cerebellar atrophy with clefts in the bilateral lobes (Figures 2A–H). DTI series demonstrated sparse and reduced right cerebral white matter fiber tracts compared to the left (Figures 2I–L). EEG showed a slight increase in medium-amplitude sharp waves and sharp slow complex waves in the right parietal region during sleep (Figure 3). ECG, ambulatory blood pressure monitoring, and echocardiography results were all within normal ranges.

Pedigree analysis indicated that the proband (IV-3) is a descendant of a consanguineous marriage as his paternal grandfather (II-1) and maternal grandmother (II-4) were siblings, resulting in his parents being first cousins. His two elder sisters are phenotypically normal. Considering the health status of other family members, we hypothesized an autosomal recessive inheritance pattern.

WES of the DNA of the proband revealed a novel homozygous variant c.4154-6C > G in the WDR62 gene (NM_001083961.2). This allele frequency of the variant was exceedingly rare, recorded at 0.000007970 in the gnomAD database.² Splice AI analysis³ indicated a high delta score of 0.96 for acceptor gain. This mutation was neither listed in ClinVar⁴ or HGMD⁵ nor reported in other major databases such as Exome Variant Server, 1,000 Genomes, or dbSNP. Sanger sequencing confirmed segregation of this mutation within the family, showing that the parents of the proband and one elder sister were healthy carriers, while the eldest sister exhibited no mutation at this site (Figure 4A).

We used MaxEntScan (20) to predict the strength of the human 5′ splice site and evaluated the c.4154-6C > G substitution. This tool rated the mutation as moderately pathogenic with a score of 6.1637. Furthermore, the dbNSFP database confirmed its pathogenicity, showing an ADA score of 0.9992 (21, 22). To understand the impact of this splice-site mutation further, we amplified and analyzed cDNA from both a negative control and the proband. Gel electrophoresis displayed a single band of approximately 493 base pairs (bp) in each sample. Nonetheless, Sanger sequencing revealed that the c.4154-6C > G mutation led to an abnormal 498-bp transcript due to the retention of 5 bp (c.4154–1 to c.4154–5 CCCAG), which was not present in the negative control (Figures 4B,C). This abnormal transcript included a premature termination codon, adding evidence to its pathogenic nature. When comparing the mutation p.Gly1385Alafs*32-truncated protein (mut-WDR62) with the wild-type (wt) WDR62 protein, we observed a significant reduction in the stability of the mutant protein (p < 0.0008,***). This reduction likely results from the unstable degradation of the truncated protein post-mutation (Figures 4D,E).