Wei Zhou

Background: SH2B1 encodes an adaptor protein involved in metabolic regulation and body‐weight control. Although SH2B1 haploinsufficiency is a recognized contributor to obesity in 16p11.2 deletion syndrome, the relationship between rare monoallelic SH2B1 variants and neurodevelopmental disorders (NDDs) remains poorly characterized. Methods: We analyzed genetic and clinical data from a cohort of individuals with NDDs. The identified rare SH2B1 variants were evaluated using bioinformatic tools and structural modeling. Functional consequences were assessed in vitro through analyses of protein expression, subcellular localization, and extracellular signal‐regulated kinase (ERK) pathway activation via EGF stimulation assays. Results: Five individuals carrying rare monoallelic SH2B1 variants presented with syndromic NDDs, including developmental delay, intellectual disability, autism spectrum disorder, dysmorphic features, and hyperphagia. Whole‐exome sequencing identified five heterozygous SH2B1 variants, including three missense variants (c.52C>G, p.Leu18Val; c.696A>T, p.Arg232Ser; c.1141C>T, p.Pro381Ser) and two truncating variants (c.90_91delTG, p.Cys30Ter; c.121_130dup, p.Arg44LeufsTer30); four of these arose de novo , whereas c.696A>T (p.Arg232Ser) was maternally inherited. The individual carrying the maternally inherited p.Arg232Ser variant presented with elevated BMI and hyperphagia. In overexpression assays, truncating variants showed reduced protein expression and abnormal subcellular localization, whereas missense variants retained comparable protein levels but showed increased EGF‐induced ERK phosphorylation in vitro, suggesting altered ERK signaling responses. Conclusions: This study further delineates the clinical spectrum associated with rare monoallelic SH2B1 variants in individuals with NDDs. Functional analyses suggest variant‐specific effects on protein expression, subcellular localization, and ERK signaling responses. These findings support the consideration of SH2B1 in genetic analyses of NDDs, particularly in individuals with co‐occurring metabolic abnormalities.

Background: Congenital anomalies of the kidney and urinary tract (CAKUT) are clinically heterogeneous and remain genetically unexplained in many patients. Biallelic variants in CTU2 have been reported in DREAM‐PL syndrome, a severe multisystem disorder characterized by dysmorphic facies, renal agenesis, ambiguous genitalia, microcephaly, polydactyly, and lissencephaly. However, to date, there have been no reports on the involvement of CTU2 in CAKUT. Methods: Exome sequencing (ES) was performed in 200 patients with CAKUT. Candidate CTU2 variants were validated by Sanger sequencing, and segregation analysis was conducted in available family members. Variant rarity was assessed using public population databases and an in‐house cohort of 200 ethnically matched kidney disease‐free controls. Potential functional effects were evaluated using in silico prediction tools and minigene splicing assays . Results: The affected individual, who had a diagnosis of CAKUT, presented with bilateral hydronephrosis, chronic kidney disease, and focal segmental glomerulosclerosis. Genetic analysis revealed compound heterozygous CTU2 variants, NM_001012759.3: c.913C>T, p.(Arg305Trp), and c.1492C>G, p.(Gln498Glu), which were inherited from his father and mother, respectively. Both variants were rare and had not been previously reported in CTU2 ‐related disease. In silico splicing analyses suggested that both variants may alter putative exonic splicing enhancers (ESE) motifs. Minigene splicing assays showed that c.913C>T altered pre‐mRNA splicing by increasing exon 9 skipping, whereas c.1492C>G had no statistically significant effect on exon inclusion. Conclusion: This study identifies two rare CTU2 variants in an individual with renal‐predominant CAKUT and provides in vitro evidence that c.913C>T partially alters splicing. While these findings do not establish CTU2 as a definitive cause of isolated CAKUT, they support further case collection and kidney‐relevant functional studies of CTU2 in renal developmental phenotypes.

Craniosynostosis is a genetically heterogeneous craniofacial disorder caused by the premature fusion of one or more cranial sutures. Pathogenic variants in

Background: Craniosynostosis (CS), a heterogeneous craniofacial disorder caused by premature fusion of cranial sutures, is sub‐classified anatomically by suture involvement (sagittal, metopic, coronal, lambdoid) and phenotypically into isolated/non‐syndromic forms or syndromic (CS with extracranial anomalies). Pathogenic variants in multiple genetic loci have been implicated in CS, with particular significance attributed to allelic variants in IL11RA (interleukin‐11 receptor alpha subunit; OMIM#600939). Clinical observations of individuals with IL11RA mutations indicate syndromic CS, characterized by dental anomalies and Crouzon‐like facial features.Methods: Genetic analyses were carried out utilizing whole‐exome sequencing, with subsequent validation through direct Sanger sequencing. IL11RA biallelic pathogenic variants were detected and further analyzed by multiple in silico prediction tools, including 3D protein modeling.Results: Our cohort comprises six pediatric patients presenting with CS linked to biallelic pathogenic mutations in IL11RA, including two previously unreported variants (p.Pro218Argfs*140, p.Trp132Ter). Three‐dimensional protein structure modeling and molecular docking simulations demonstrated that four missense variants (p.Pro116Leu, p.Glu126Gly, p.Gly231Val, p.Leu236Pro) disrupt hydrogen bond networks critical for maintaining the IL‐11 receptor alpha subunit's tertiary structure, significantly reducing ligand‐binding affinity to both interleukin‐11 (IL‐11) and gp130.Conclusion: This study describes the clinical phenotype of six children with craniosynostosis and reveals novel variants in the IL11RA gene, thereby broadening the genotypic spectrum associated with this gene. Given the scarcity of patients reported in the literature, a detailed examination of the specific clinical and molecular characteristics will benefit our understanding of craniosynostosis caused by IL11RA variants.

Brain malformations with or without urinary tract defects (BRMUTD) are caused by heterozygous variants in the NFIA gene. BRMUTD is a neurodevelopmental disorder characterized by hypoplasia or absence of the corpus callosum, hydrocephalus or ventriculomegaly, and developmental delay, which may or may not be accompanied by urinary tract defects. Here, we report the successful generation of induced pluripotent stem cells (hiPSCs) from a 3-year-old male BRMUTD patient using Sendai virus-based non-integrating reprogramming technology. This patient-derived cell line harbors an intragenic deletion within the NFIA gene (NC_000001.10: g.61650967_61842967del [GRCh37]), which is associated with a significant reduction in NFIA expression. This cell line maintains a normal karyotype, expresses pluripotency markers, and can differentiate into three germ layers. The established hiPSCs line will provide an in vitro model for studying pathological mechanisms and potential therapies of NFIA-related neurodevelopmental disorder.

(1) Background: Autosomal recessive polycystic kidney disease (ARPKD) is a rare ciliopathy characterized by progressively enlarged kidneys with fusiform dilatation of the collecting ducts. Loss-of-function mutations in the PKHD1 gene, which encodes fibrocystin/polyductin, cause ARPKD; however, an efficient treatment method and drug for ARPKD have yet to be found. Antisense oligonucleotides (ASOs) are short special oligonucleotides which function to regulate gene expression and alter mRNA splicing. Several ASOs have been approved by the FDA for the treatment of genetic disorders, and many are progressing at present. We designed ASOs to verify whether ASOs mediate the correction of splicing further to treat ARPKD arising from splicing defects and explored them as a potential treatment option. (2) Methods: We screened 38 children with polycystic kidney disease for gene detection using whole-exome sequencing (WES) and targeted next-generation sequencing. Their clinical information was investigated and followed up. The PKHD1 variants were summarized and analyzed, and association analysis was carried out to analyze the relationship between genotype and phenotype. Various bioinformatics tools were used to predict pathogenicity. Hybrid minigene analysis was performed as part of the functional splicing analysis. Moreover, the de novo protein synthesis inhibitor cycloheximide was selected to verify the degraded pathway of abnormal pre-mRNAs. ASOs were designed to rescue aberrant splicing, and this was verified. (3) Results: Of the 11 patients with PKHD1 variants, all of them exhibited variable levels of complications of the liver and kidneys. We found that patients with truncating variants and variants in certain regions had a more severe phenotype. Two splicing variants of the PKHD1 genotypes were studied via the hybrid minigene assay: variants c.2141-3T>C and c.11174+5G>A. These cause aberrant splicing, and their strong pathogenicity was confirmed. We demonstrated that the abnormal pre-mRNAs produced from the variants escaped from the NMD pathway with the use of the de novo protein synthesis inhibitor cycloheximide. Moreover, we found that the splicing defects were rescued by using ASOs, which efficiently induced the exclusion of pseudoexons. (4) Conclusion: Patients with truncating variants and variants in certain regions had a more severe phenotype. ASOs are a potential drug for treating ARPKD patients harboring splicing mutations of the PKHD1 gene by correcting the splicing defects and increasing the expression of the normal PKHD1 gene.

Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) are characterized by acute lung inflammation. Intercellular adhesion molecule-1 (ICAM-1) and interleukin-8 (IL-8) play an important role in the development of these diseases. Mitogen-activated protein kinase (MAPK) p38/activated protein kinase 2 (MK2) regulates the expression of ICAM-1 and IL-8 in human lung microvascular endothelial cells (HPMECs) stimulated by tumor necrosis factor-α (TNF-α); however, the underlying molecular mechanism remains unclear. Here, we show that human antigen R (HuR), an RNA binding protein which binds preferentially to AU-rich elements (AREs) and stabilizes mRNAs, regulates TNF-α-induced ICAM-1 expression in the MK2/HuR signaling pathway. MK2 and HuR were silenced respectively in HPMECs and then HPMECs were stimulatied with TNF-α. Nucleo-cytoplasmic shuttling of HuR was detected by subcellular fractionation and confocal microscopy in MK2 knockdown HPMECs. In HuR silencing cells, protein and mRNA levels of ICAM-1 and IL-8 were measured by western blot analysis, ELISA and real-time PCR; mRNA stabilization were measured by real-time PCR after actinomycin D (ActD) blocking transcription. Furthermore, we performed neutrophil adhesion assay to assess the adhering capacity after HuR silencing. MK2 were subjected to a knockdown by interfering RNA, the mRNA and protein levels of HuR in human pulmonary microvascular endothelial cells (HPMECs) were not affected. However, after the stimulation of TNF-α, silencing MK2 inhibited HuR accumulation to cytoplasm from nucleus in HPMECs. Consequently, knockdown of HuR by RNA interference in HPMECs, there was reduction in the stability of ICAM-1 mRNA and ICAM-1 protein level. This event was accompanied by a decrease in the adhesion of neutrophils towards HPMECs. Nevertheless, HuR silencing had no effect on the mRNA and protein levels of IL-8. These results indicate that MK2 post-transcriptionally regulates TNF-α-induced ICAM-1 expression by altering the cytoplasmic localization of HuR in HPMECs.