Yu An

Synonymous mutations, once regarded as silent, are increasingly recognized as pathogenic through disruption of mRNA splicing. Here, we report a prenatal case with classic features of Smith-Lemli-Opitz syndrome (SLOS), including increased nuchal translucency, enlarged kidneys, and fetal growth restriction. Whole-exome sequencing identified compound heterozygous DHCR7 variants: a previously reported missense variant, NM_001360.3:c.1220A > G (p.Asn407Ser), and a novel synonymous variant, NM_001360.3:c.963G > A (p.Gln321 =). Functional validation using minigene assays and patient-derived cDNA demonstrated that c.963G > A causes aberrant splicing, resulting in a premature termination codon and production of a truncated protein lacking the sterol-sensing domain, which contains multiple recurrent pathogenic missense sites. These findings establish the deleterious effect of this synonymous change, analogous to the well-characterized splice-site variant NM_001360.3:c.964 − 1G > C (historically referred to as IVS8 − 1G > C). In sum, this study provides the first definitive evidence that a synonymous DHCR7 variant can act as a likely pathogenic allele through splicing disruption. Moreover, it offers critical molecular insight and a refined framework for interpreting synonymous variants—particularly variants of uncertain significance—with important implications for clinical diagnosis, genetic counseling, and prenatal care.

Our earlier human studies identified NRSN2 (Neurensin-2), a neuronal-specific vesicular protein, as a candidate gene contributing to 20p13 microdeletion syndrome, yet the functional consequences of NRSN2 deficiency in the nervous system remain poorly understood. To explore the role of Nrsn2 in neurodevelopment and cognitive function, we utilized previously generated homozygous Nrsn2 knockout mice (Nrsn2^-/-) and performed a series of behavioral, morphological, and electrophysiological analyses. Behaviorally, Nrsn2^-/- mice exhibited mild locomotor impairment, as assessed by gait analysis at 4 and 8 weeks of age, as well as significant deficits in spatial learning and memory (Morris water maze) and fear memory (passive avoidance test) at 8 weeks. Morphometric analysis suggested no overt alterations in dendritic complexity or spine density in hippocampal CA1 pyramidal neurons or cerebellar Purkinje cells without developmental malformation. Electrophysiological recordings and immunoblotting analyses may reflect region-specific synaptic alterations. In the hippocampus, expression levels of the NMDA receptor subunits GluN1 and GluN2A were reduced at 4 weeks of age. Consistently, CA1 pyramidal neurons displayed decreased sEPSC frequency with unchanged amplitude under the conditions examined. In addition, an imbalance in hippocampal excitatory/inhibitory transmission was observed, as reflected by altered sEPSC frequency in the absence of changes in sIPSC frequency. In cerebellar Purkinje cells, GluA1-containing AMPA receptors were selectively downregulated, accompanied by reduced frequency and amplitude of sEPSCs and a selective decrease in sIPSC frequency, indicating both excitatory and inhibitory synaptic dysfunction in this region. Collectively, these findings indicate that Nrsn2 deficiency is accompanied by altered excitatory synaptic transmission and reduced long-term potentiation (LTP) at 8 weeks of age, despite preserved dendritic architecture as assessed by Golgi staining. These synaptic and plasticity deficits occur alongside the observed cognitive and motor impairments in Nrsn2^⁻/⁻ mice. This study provides a descriptive phenotypic characterization of Nrsn2 deficiency and offers initial insights into the neurobiological role of NRSN2 and its contribution to neurodevelopment, learning, and memory.

Objective: Drug‐resistant epilepsy (DRE) affects approximately one‐third of patients with epilepsy. The molecular heterogeneity underlying DRE remains poorly defined, largely due to limited access to resected brain tissue and substantial genetic diversity. Current classifications rely primarily on clinical symptoms and histopathological features rather than molecular mechanisms, constraining mechanistic insight and the development of targeted therapies. This study aimed to develop a transcriptome‐based, machine learning–guided framework for molecular classification of DRE. Methods: We performed comprehensive RNA sequencing on 153 surgically resected samples from 95 patients with DRE. Two transcriptomic subtypes were identified through unsupervised clustering. We also leveraged a weighted correlation network‐based framework and systematic transcriptional signature comparison and developed a classification model using machine learning algorithms. Results: Unsupervised clustering revealed two molecular subtypes that diverged from traditional pathological classifications, indicating an alternative transcriptomic basis for epilepsy pathogenesis. A classification model was constructed based on four key differentially regulated pathways: (1) neuroactive ligand–receptor interaction, (2) cAMP signaling, (3) γ‐aminobutyric acid (GABA)ergic synapse, and (4) calcium signaling. Among the tested algorithms, the random forest model demonstrated superior performance, achieving 96% classification accuracy with an area under the curve (AUC) of .95. Significance: These molecular subtypes and their pathways could serve as key molecular hallmarks of epilepsy, offering valuable insights for developing targeted therapies. Moreover, our findings introduce a novel framework for classifying epilepsy based on its molecular nature, potentially connecting the clinical symptoms with the underlying causes more effectively.

Background Parkinson’s Disease (PD) diagnosis lacks effective non-invasive markers, complicating early detection and timely intervention. Machine learning (ML) combined with clinical blood biomarkers may provide a feasible approach for early diagnosis and monitoring. Aim This study aims to construct and validate a non-invasive diagnostic model for PD using machine learning and routine clinical blood biomarkers, and identify key biomarkers linked to disease severity. Methods A total of 920 participants (428 PD and 492 non-PD) from two medical centers were included as training and validation sets. Biomarker selection was performed via least absolute shrinkage and selection operator (LASSO) and stepwise regression. Five machine learning models—logistic regression (LR), support vector machine (SVM), decision tree (DT), Naive Bayes (NB) and K-Nearest Neighbor (KNN)—were constructed and compared. The optimal model was interpreted using Shapley values (SHAP), and correlation with PD severity (Hoehn-Yahr stage) was assessed. Results The SVM model demonstrated the best external validation performance (AUC = 0.916, recall = 0.949, F1-score = 0.843). SHAP analysis revealed superoxide dismutase (SOD) contributed the most to the model prediction, followed by gender and uric acid (UA). Furthermore, albumin (ALB) and SOD showed significant negative correlations with PD severity. Conclusion The SVM-based diagnostic model effectively differentiates PD from controls using readily obtainable clinical biomarkers, offering promising clinical utility for PD screening, diagnosis, and progression monitoring.

β-propeller protein-associated neurodegeneration (BPAN) is characterized by global developmental delay, intellectual disability, and epileptic encephalopathies in infancy or early childhood caused by WDR45/WIPI4 gene mutations. WDR45 depletion disrupted autophagy, leading to iron accumulation in the brain and contributing to neuronal apoptosis. The impact on neuron performance remains unknown. Our previous study established the iPSC cell line derived from a girl patient with a de novo variant c.344 + 5G > T in WDR45 (FDHPIi001). This study demonstrated that this intron 6 mutation impairs RNA splicing, resulting in a 28 bp insertion and nonsense-mediated mRNA decay (NMD) of truncated WDR45. Upon differentiating the iPSCs into dopaminergic neurons, we observed significantly shorter neuronal axons using high-intensity imaging analysis. Additionally, there was significant ferritin accumulation in the induced neurons but not in the iPSCs from the same patient. This research has elucidated the pathogenicity of a non-canonical splice site mutation in WDR45 and has provided deeper insights into the pathologies of neurodegenerative diseases caused by WDR45 defects.

Accurate variant calling is essential for next-generation sequencing (NGS)-based diagnosis of rare diseases, yet most benchmarking studies have focused on standard cell lines or trio-based samples, with limited relevance to sporadic cases. Here, we systematically compared the performance of DeepVariant and GATK HaplotypeCaller in two Chinese cohorts of patients with sporadic epilepsy (EP) and autism spectrum disorder (ASD). DeepVariant exhibited higher precision and sensitivity in detecting single nucleotide variants (SNVs), while GATK showed a distinct advantage in identifying rare variants, which are often key to understanding the genetic basis of rare diseases. Comparative analyses based on disease-related gene panels further highlighted differences in the identification of potentially deleterious variants. These findings reveal important trade-offs between variant callers and emphasize the need to tailor variant-calling strategies to specific research and clinical contexts. Our study provides practical vision for optimizing germline variant detection pipelines in sporadic neurodevelopmental disorders, offering broader insights for precision medicine applications.

Our earlier human studies identified NRSN2 (Neurensin-2), a neuronal-specific vesicular protein, as a candidate gene contributing to 20p13 microdeletion syndrome, yet the mechanisms linking NRSN2 deficiency to the core phenotypes remain unknown. To elucidate the function of Nrsn2 in neurodevelopment and cognitive function, we generated homozygous Nrsn2 knockout mice (Nrsn2⁻/⁻) and performed a series of behavioral, morphological, and electrophysiological analyses. Behaviorally, Nrsn2⁻/⁻ mice exhibited significant impairments in spatial learning and memory (Morris water maze) and fear memory (passive avoidance test). Morphometric analysis revealed no alterations in dendritic complexity or spine density in hippocampal CA1 pyramidal neurons or cerebellar Purkinje cells, ruling out developmental malformation. Electrophysiology and immunoblotting uncovered region-specific synaptic alterations. Collectively, Nrsn2 deficiency does not affect basal synaptic transmission through presynaptic release probability (PRP) but impairs excitatory synaptic transmission via downregulation of GluN1- and GluN2A-containing NMDA receptors and causes a frequency-specific decrease in sEPSCs amplitude in the hippocampus, as well as impaired hippocampal synaptic plasticity, evidenced by reduced long-term potentiation (LTP). In addition, we observed the disruption of hippocampal excitatory/inhibitory balance, indicated by altered sEPSC frequency but unchanged sIPSC frequency. In cerebellar Purkinje cells, GluA1-containing AMPA receptors were downregulated, accompanied by reduced frequency and amplitude of sEPSCs and a selective reduction in sIPSCs frequency. These findings demonstrate that NRSN2 is essential for maintaining synaptic receptor composition and functional plasticity, particularly within excitatory circuits. Consequently, NRSN2 deficiency disrupts synaptic transmission, rather than structural neuronal integrity, and likely underlies the observed cognitive and motor phenotypes. This study provides novel mechanistic insights into the neurobiological role of NRSN2 and its contribution to neurodevelopment, learning, and memory.

Neurensin-2 (Nrsn2) is a neuro-specific gene linked to neurodevelopmental disorders and has recently been reported to function as a bidirectional emotional regulator, highlighting its molecular roles in the nervous system. However, the connections between Nrsn2, brain architecture, and functionality remain to be fully elucidated. Our study utilized 11.7 T multimodal magnetic resonance imaging (MRI) to assess the impact of Nrsn2 gene knockout on the brain’s microstructure, regional functional activity, and network connectivity during different developmental phases in mice. We observed significant changes in the functional brain network connectivity of Nrsn2^−/− mice without marked differences in brain microstructure or regional activity. These changes were particularly pronounced in sensory-related areas, such as the gustatory and auditory systems, in both juvenile and adult specimens. Previous studies have correlated the enhanced Default Mode Network (DMN) with depression, and Nrsn2 knockout has been associated with stress resilience. Our findings further revealed reduced connectivity in various DMN regions in adult Nrsn2^−/− mice, suggesting a potential link to increased stress tolerance. Moreover, the sensory system’s critical role in environmental perception implies that alterations in network connectivity due to Nrsn2 knockout could affect the processing and integration of external inputs, thereby influencing emotional experiences.

To explore the genetic basis for a pregnant woman with a history of adverse pregnancy outcomes. A woman with an adverse history of pregnancies including one fetal demise and two induced abortions due to fetal diaphragmatic hernia and complex cardiac anomalies was selected as the study subject. Muscle tissue from the induced abortus was subjected to whole exome sequencing, and candidate variant was verified by Sanger sequencing of the couple and other family members. Genetic sequencing revealed that the fetus has harbored a frameshift variant of the KDM6A gene (NM_001291415.2), namely c.1228_1229del (p.Gln410GlufsTer2), which was inherited from the woman and her mother. The variant was unreported previously, and the woman was found to have short stature, sparse eyebrows in the outer third, peculiar facial features, but normal intelligence in addition with female congenital genital malformation, like incomplete vaginal septum, double cervix, double uterus, and unilateral ovary absence. mostly similar phenotypes observed in her mother. The hemizygous c.1228_1229del variant of the KDM6A gene probably underlay the abnormalities in the fetus. All findings have enabled genetic counseling for this family featuring X-linked inheritance, and the woman had given birth to a healthy girl with appropriate prevention and intervention.

So far, over 20 causative genes of monogenic Parkinson’s disease (PD) have been identified. Some causative genes of non-parkinsonian entities may also manifest with parkinsonism mimicking PD. This study aimed to investigate the genetic characteristics of clinically diagnosed PD with early onset age or family history. A total of 832 patients initially diagnosed with PD were enrolled, of which, 636 were classified into the early-onset group and 196 were classified into the familial late-onset group. The genetic testing included the multiplex ligation-dependent probe amplification and next generation sequencing (target sequencing or whole-exome sequencing). The dynamic variants of spinocerebellar ataxia were tested in probands with family history. In the early-onset group, 30.03% of patients (191/636) harbored pathogenic/likely pathogenic (P/LP) variants in known PD-related genes (CHCHD2, DJ-1, GBA (heterozygous), LRRK2, PINK1, PRKN, PLA2G6, SNCA and VPS35). Variants in PRKN were the most prevalent, accounting for 15.72% of the early-onset patients, followed by GBA (10.22%), and PLA2G6 (1.89%). And 2.52% (16/636) had P/LP variants in causative genes of other diseases (ATXN3, ATXN2, GCH1, TH, MAPT, GBA (homozygous)). In the familial late-onset group, 8.67% of patients (17/196) carried P/LP variants in known PD-related genes (GBA (heterozygous), HTRA2, SNCA) and 2.04% (4/196) had P/LP variants in other genes (ATXN2, PSEN1, DCTN1). Heterozygous GBA variants (7.14%) were the most common genetic cause found in familial late-onset patients. Genetic testing is of vital importance in differential diagnosis especially in early-onset and familial PD. Our findings may also provide some clues to the nomenclature of genetic movement disorders.