OBJECTIVES: Uridine diphospho-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase (GNE) myopathy is a progressive neurodegenerative disease associated with homozygous or compound heterozygous missense mutations in the GNE gene. This study aims to explore the impact of the homozygous p.V543M mutation in on cell phenotype and to gain preliminary insights into the underlying mechanisms. METHODS: Human embryonic kidney 293T (HEK 293T) cells were used to construct wild-type (WT-GNE) and mutant (MUT-GNE) GNE overexpression models. Western blotting and immunofluorescence were used to assess GNE protein expression levels and subcellular localization. Cell adhesion, proliferation, apoptosis, and mitochondrial membrane potential were evaluated using the cell counting kit-8 (CCK-8) assay, crystal violet staining, flow cytometry, Hoechst 33342/propidium iodide (PI) staining, and tetramethylrhodamine ethyl ester (TMRE) staining. Sialic acid synthesis levels and GNE enzymatic activity were measured, and the mRNA expression of sialic acid biosynthesis-related enzymes was quantified by real-time PCR. RESULTS: Western blotting confirmed successful establishment of GNE overexpression models. Immunofluorescence showed significantly reduced co-localization of GNE protein with Golgin-97 in the MUT-GNE group compared to WT-GNE (Pearson's correlation coefficient: 0.65±0.08 vs 0.83±0.06, P<0.05). Compared with WT-GNE, cells in the MUT-GNE group exhibited increased adhesion, decreased proliferation, and reduced mitochondrial membrane potential (P<0.05). No significant differences in apoptosis were observed between groups. The MUT-GNE group showed reduced sialic acid production, significantly decreased kinase activity, and downregulated transcription of sialic acid biosynthesis-related enzymes compared to WT-GNE (P<0.001). CONCLUSIONS The p.V543M mutation in the GNE gene alters cellular phenotype by reducing GNE enzymatic activity and the transcription of sialic acid biosynthesis enzymes, ultimately impairing sialic acid production.
Humans ; Mutation, Missense ; HEK293 Cells ; Apoptosis/genetics* ; Phenotype ; Multienzyme Complexes/metabolism* ; Cell Proliferation ; Homozygote ; Cell Adhesion/genetics* ; Distal Myopathies/genetics*

Autosomal dominant tubulointerstitial kidney disease (ADTKD) is a rare autosomal dominant hereditary disorder characterized by hyperuricemia, gout, impaired urinary concentration, and progressive renal failure. It is primarily caused by mutations in uromodulin (UMOD) gene. This study reports a family with ADTKD in which whole-exome sequencing and Sanger sequencing identified a missense mutation in the UMOD gene, c.761A>C (p.H254P), present in both the proband and affected relatives. According to American College of Medical Genetics and Genomics (ACMG) guidelines, this variant is classified as likely pathogenic. The mutation results in an amino acid substitution that may impair UMOD protein folding and intracellular trafficking. UMOD gene mutations are associated with ADTKD, and genetic testing plays a vital role in the early diagnosis and treatment of this condition, highlighting its importance in the diagnosis of rare kidney diseases.
Adult ; Humans ; Male ; Exome Sequencing ; Mutation ; Mutation, Missense ; Nephritis, Interstitial/genetics* ; Pedigree ; Uromodulin/genetics*

OBJECTIVES: Keratoconus (KC) is a progressive corneal ectasia disorder, arising from a myriad of causes including genetic predispositions, environmental factors, biomechanical influences, and inflammatory reactions. This study aims to identify potential pathogenetic gene mutations in patients with sporadic KC in the Han Chinese population. METHODS: Twenty-five patients with primary KC as well as 50 unrelated population-matched healthy controls, were included in this study to identify potential pathogenic gene mutations among sporadic KC patients in the Han Chinese population. Sanger sequencing and whole-exome sequencing (WES) were used to analyze mutations in the zinc finger protein 469 (ZNF469) gene. Bioinformatics analysis was conducted to explore the potential role of ZNF469 in KC pathogenesis. RESULTS: Five novel heterozygous missense variants were identified in KC patients. Among them, 2 compound heterozygous variants, c.8986G>C (p. E2996Q) with c.11765A>C (p. D3922A), and c.4423C>G (p. L1475V) with c.10633G>A (p. G3545R), were determined to be possible pathogenic factors for KC. CONCLUSIONS Mutations in the ZNF469 gene may contribute to the development of KC in the Han Chinese population. These mutation sites may provide valuable information for future genetic screening of KC patients and their families.
Adolescent ; Adult ; Female ; Humans ; Male ; Case-Control Studies ; China/ethnology* ; Exome Sequencing ; Genetic Predisposition to Disease ; Keratoconus/genetics* ; Mutation ; Mutation, Missense ; Transcription Factors/genetics* ; East Asian People/genetics*

The KCNQ potassium channels play a crucial role in modulating neural excitability, and their dysfunction is closely associated with epileptic disorders. While variants in KCNQ2 have been extensively studied, KCNQ3-related disorders have rarely been reported. With advances in next-generation sequencing technologies, an increasing number of cases of KCNQ3-related disorders have been identified. However, the correlation between genotype and phenotype remains poorly understood. In this study, we established a variant library consisting of 24 missense mutations in KCNQ3 and introduced these mutations into three different template types: KCNQ3, KCNQ3-A315T (Q3*), and KCNQ3-KCNQ2 tandem (Q3-Q2). We then analyzed the effects of these mutations on the KCNQ3 channel function using patch-clamp recording. The most informative parameter across all three backgrounds was the current density of the mutant channels. The current density patterns in the Q3* and Q3-Q2 backgrounds were similar, with most mutations resulting in an almost complete loss of function (LOF), they were concentrated in the pore-forming domain of KCNQ3. In contrast, mutations in the voltage-sensing domain or C-terminus did not show significant differences from the wild-type channel. Interestingly, these LOF mutations were typically associated with self-limited familial neonatal epilepsy, while neurodevelopmental disorders (NDD) were more closely associated with mutations that did not significantly differ from the wild-type. V_1/2, another important parameter of the electrophysiological properties, could not be accurately determined in the majority of KCNQ3 mutations due to its nearly complete LOF in the Q3* and Q3-Q2 backgrounds. Intriguingly, the V_1/2 of functional mutations were primarily leftward shifted, indicating a gain-of-function (GOF) effect, which was typically associated with NDD. In addition to previously reported mutations, we identified G553R as a novel GOF mutation. In the co-transfection background, parameters such as V_1/2 could be determined, but the dysfunctional effects of these mutations were mitigated by the co-expression of wild-type KCNQ3 and KCNQ2 subunits, resulting in no significant differences between most mutations and the wild-type channel. Furthermore, we applied KCNQ modulators to reverse the electrophysiological abnormalities caused by KCNQ3 variants. The LOF mutations were reversed by the application of Pynegabine (HN37), a KCNQ opener, while the GOF mutation responded well to Amitriptyline (AMI), a KCNQ inhibitor. These findings provide essential insights into the pathogenic mechanisms underlying KCNQ3-related disorders and may inform clinical decision-making.
KCNQ3 Potassium Channel/genetics* ; Humans ; Mutation, Missense/genetics* ; KCNQ2 Potassium Channel/genetics* ; Patch-Clamp Techniques ; HEK293 Cells ; Animals ; Phenylenediamines/pharmacology* ; Carbamates

Muenke syndrome is an autosomal dominant genetic disorder that is typically characterized by unilateral or bilateral coronal synostosis, macrocephaly, midface hypoplasia, and developmental delays. This article reports a case of Muenke syndrome with a soft cleft palate. A heterozygous missense mutation c.749C>G (p.P250A) was identified in the FGFR3 gene through genetic testing. The patient exhibited typical features including coronal synostosis, bilateral hearing loss, right accessory auricle, and developmental delays and underwent surgery to repair the soft cleft palate. Cases of Muenke syndrome with cleft palate in the literature are relatively rare, and common associated symptoms include coronal suture craniosynostosis and hearing impairment. This article reports a differential diagnosis with other craniosynostosis syndromes and provides a reference for clinical diagnosis and treatment.
Humans ; Cleft Palate/surgery* ; Craniosynostoses/diagnosis* ; Mutation, Missense ; Palate, Soft/abnormalities* ; Receptor, Fibroblast Growth Factor, Type 3/genetics*

Collagen, a vital matrix protein for various tissue and functions in animals, is widely applied in biomaterials. In type Ⅰ collagen, missense mutations of glycine (Gly) in the Gly-Xaa-Yaa triplet of the triple helix are a major cause of osteogenesis imperfecta (OI). Clinical manifestations exhibit marked heterogeneity, spanning a broad disease spectrum from mild skeletal fragility (Type Ⅰ) to severe limb deformities (Type Ⅲ) and perinatal lethal forms (Type Ⅱ). This study utilized recombinant collagen as a model to further elucidate whether Gly→Ala/Val mutations at the N-terminus of the integrin-binding sequence GFPGER affect collagen structure and function, and to explore the underlying mechanisms by which missense mutations impact the biological function of collagen. By introducing Ala and Val substitutions at seven Gly positions N-terminal to the GFPGER sequence, we systematically assessed the effects of these amino acid replacements on the triple-helical structure, thermal stability, integrin-binding ability, and cell adhesion of recombinant collagen. All constructs formed a stable triple-helix structure, with slightly compromised thermal stability. Gly→Val substitutions increased the susceptibility of recombinant collagen to trypsin, which suggested local conformational perturbations in the triple helix. In addition, Gly→Val substitutions significantly reduced the integrin-binding affinity and decreased HT1080 cell adhesion, with the effects stronger than Gly→Ala substitutions. Compared with Gly→Ala substitutions, substitution of Gly with the larger residue Val had enhanced negative effects on the structure and function of recombinant collagen. These findings provide new insights into the molecular mechanisms of osteogenesis imperfecta and offer theoretical references and experimental foundations for the design of collagen sequences and the development of collagen-based biomaterials.
Recombinant Proteins/biosynthesis* ; Glycine/genetics* ; Humans ; Osteogenesis Imperfecta/genetics* ; Integrins/metabolism* ; Collagen/metabolism* ; Collagen Type I/metabolism* ; Amino Acid Substitution ; Mutation ; Mutation, Missense

Male infertility is a worldwide health issue, affecting 8%-12% of the global population. Oligoasthenoteratozoospermia (OAT) represents a severe type of male infertility, characterized by reduced sperm count and motility and an increased frequency of sperm with aberrant morphology. Using whole-exome sequencing, this study identified a novel missense mutation (c.848C>A, p.A283E) in the coiled-coil domain-containing 34 gene (CCDC34) in a consanguineous Pakistani family. This rare mutation was predicted to be deleterious and to affect the protein stability. Hematoxylin and eosin staining of spermatozoa from the patient with OAT revealed multiple morphological abnormalities of the flagella and transmission electron microscopy indicated axonemal ultrastructural defects with a lack of outer dynein arms. These findings indicated that CCDC34 plays a role in maintaining the axonemal ultrastructure and the assembly or stability of the outer dynein arms, thus expanding the phenotypic spectrum of CCDC34 missense mutations.
Humans ; Male ; Mutation, Missense/genetics* ; Pakistan ; Consanguinity ; Asthenozoospermia/genetics* ; Pedigree ; Infertility, Male/genetics* ; Adult ; Oligospermia/genetics* ; Exome Sequencing ; Axoneme/ultrastructure* ; Spermatozoa/ultrastructure*

Humans ; Mutation, Missense ; KCNQ1 Potassium Channel/genetics* ; Long QT Syndrome/genetics* ; Phenotype ; Mutation ; Pedigree

Humans ; Mutation, Missense ; KCNQ1 Potassium Channel/genetics* ; Long QT Syndrome/genetics* ; Phenotype ; Mutation ; Pedigree

Objective: To explore the relationship between pathogenic gene, mutation and phenotype of left ventricular noncompaction (LVNC) patients and their family members. Methods: The subjects were the proband with LVNC and her family members. The medical history including electrocardiogram, echocardiography and cardiac magnetic resonance examination of the proband and family members were collected. Whole exome sequencing of the proband was performed, bioinformatics analysis focused on the genes related to hereditary cardiomyopathy. Candidate pathogenic sites were validated by Sanger sequencing. The clinical interpretation of sequence variants were classified according to American College of Medical Genetics and Genomics (ACMG) guidelines. Results: The proband carried a heterozygous variation of the MYBPC3 gene c.C2827T and the MYH7 gene c.G2221C. The proband's sister carried heterozygous variation of MYBPC3 gene c.C2827T. According to the ACMG guidelines, the variant was determined to be pathogenic. Conclusion: The missense variant of MYBPC3 gene c.C2827T and MYH7 gene c.G2221C are identified from the proband with LVNC and her family member, which provides a genetic basis for clinical diagnosis and genetic counseling of the patients and the family members with LVNC.
Female ; Humans ; Cardiac Myosins/genetics* ; Heart Defects, Congenital ; Mutation ; Mutation, Missense ; Myosin Heavy Chains/genetics* ; Pedigree ; Phenotype