Primary ciliary dyskinesia (PCD) is a clinically rare, genetically and phenotypically heterogeneous condition characterized by chronic respiratory tract infections, male infertility, tympanitis, and laterality abnormalities. PCD is typically resulted from variants in genes encoding assembly or structural proteins that are indispensable for the movement of motile cilia. Here, we identified a novel nonsense mutation, c.466G>T, in cilia- and flagella-associated protein 300 ( CFAP300 ) resulting in a stop codon (p.Glu156*) through whole-exome sequencing (WES). The proband had a PCD phenotype with laterality defects and immotile sperm flagella displaying a combined loss of the inner dynein arm (IDA) and outer dynein arm (ODA). Bioinformatic programs predicted that the mutation is deleterious. Successful pregnancy was achieved through intracytoplasmic sperm injection (ICSI). Our results expand the spectrum of CFAP300 variants in PCD and provide reproductive guidance for infertile couples suffering from PCD caused by them.
Adult ; Female ; Humans ; Male ; Pregnancy ; China ; Ciliary Motility Disorders/genetics* ; Codon, Nonsense ; East Asian People/genetics* ; Exome Sequencing ; Homozygote ; Infertility, Male/genetics* ; Kartagener Syndrome/genetics* ; Pedigree ; Sperm Injections, Intracytoplasmic ; Cytoskeletal Proteins/genetics*

OBJECTIVE: To explore the genotype-phenotype relationship in a child with Opitz G/BBB syndrome (OS) with mild clinical phenotype. METHODS: A child with motor developmental delay as the initial symptom admitted to Xi'an Children's Hospital on June 10, 2021 was selected for this study. Clinical data were collected, and peripheral blood samples were obtained from the child and his mother. Whole exome sequencing (WES) was performed to identify genetic variant in the child. Candidate variant were verified by Sanger sequencing to assess inheritance patterns and pathogenicity. Real-time fluorescence quantitative PCR (RT-qPCR) and Western blot (WB) analyses were conducted to evaluate the effects of the variant on mRNA and protein expression, respectively, using recombinant expression plasmids generated in vitro. This study was approved by the Medical Ethics Committee of Xi'an Children's Hospital (Ethics No. 20240045). RESULTS: The child, a 9-month-and-7-day-old boy, presented with a low nasal bridge, hypertelorism, and difficulty sitting independently. Echocardiography revealed an atrial septal defect. WES identified a homozygous variant in the MIDI gene, c.1483C>T (p.R495X), which was confirmed by Sanger sequencing and found to be inherited from the mother.Recombinant expression plasmids were successfully constructed. RT-qPCR analysis showed that the variant significantly reduced MIDI gene mRNA expression, while WB results indicated that the variant led to the production of a truncated protein. CONCLUSION The mild clinical phenotype of OS in this child may be attributed to the mRNA degradation escape mechanism induced by the nonsense variant c.1483C>T (p.R495X) in the MIDI gene. These findings provide valuable diagnostic insights for this pedigree and contribute to the understanding of the genotype-phenotype correlation in OS.
Humans ; Male ; Infant ; Transcription Factors/metabolism* ; Microtubule Proteins/genetics* ; Craniosynostoses/genetics* ; Hypospadias/genetics* ; Codon, Nonsense/genetics* ; RNA, Messenger/metabolism* ; Female ; RNA Stability/genetics* ; Phenotype ; Nuclear Proteins/genetics* ; Ubiquitin-Protein Ligases ; Esophagus/abnormalities* ; Hypertelorism

OBJECTIVE: To carry out pathological and genetic analyses on a fetus with intrauterine growth restriction and death during second trimester after induced abortion. METHODS: A fetus undergone induced abortion due to intrauterine growth restriction and death during second trimester at the the Seventh Affiliated Hospital of Sun Yat-Sen University in 2024 was selected as the study subject. Clinical data of the pregnancy were collected. DNA was extracted from tissues from the aborted fetus and peripheral blood samples from its parents. Chromosomal microarray analysis and whole exome sequencing were carried out. Candidate variants were verified by Sanger sequencing. Following abortion, routine autopsy and pathological analysis were conducted. This study was approved by the Medical Ethics Committee of the hospital (Ethics No.: KY-2025-334-01). RESULTS: The aborted fetus was a male and harbored compound heterozygous nonsense variants of the TH gene (c.457C>T/p.Arg153* and c.694C>T/p.Gln232*), for which both parents were heterozygous carriers. Autopsy and pathological analysis revealed that the fetus had pathological features including loose arrangement of myocardial fibers and congestion in the liver. CONCLUSION Biallelic null variants of the TH gene may cause heart failure by affecting the development of cardiovascular system, which in turn may lead to intrauterine death. This study has provided new clues for the molecular diagnosis of stillbirth and recurrent pregnancy loss.
Humans ; Female ; Pregnancy ; Male ; Heterozygote ; Codon, Nonsense/genetics* ; Fetal Growth Retardation/pathology* ; Adult ; Stillbirth/genetics*

Objective: To analyze the phenotype and genotype of two pedigrees with inherited fibrinogen (Fg) deficiency caused by two heterozygous mutations. We also preliminarily probed the molecular pathogenesis. Methods: The prothrombin time (PT), activated partial thromboplastin time (APTT), thrombin time (TT) and plasma fibrinogen activity (Fg∶C) of all family members (nine people across three generations and three people across two generations) were measured by the clotting method. Fibrinogen antigen (Fg:Ag) was measured by immunoturbidimetry. Direct DNA sequencing was performed to analyze all exons, flanking sequences, and mutated sites of FGA, FGB, and FGG for all members. Thrombin-catalyzed fibrinogen polymerization was performed. ClustalX 2.1 software was used to analyze the conservatism of the mutated sites. MutationTaster, PolyPhen-2, PROVEAN, SIFT, and LRT online bioinformatics software were applied to predict pathogenicity. Swiss PDB Viewer 4.0.1 was used to analyze the changes in protein spatial structure and molecular forces before and after mutation. Results: The Fg∶C of two probands decreased (1.28 g/L and 0.98 g/L, respectively). The Fg∶Ag of proband 1 was in the normal range of 2.20 g/L, while it was decreased to 1.01 g/L in proband 2. Through genetic analysis, we identified a heterozygous missense mutation (c.293C>A; p.BβAla98Asp) in exon 2 of proband 1 and a heterozygous nonsense mutation (c.1418C>G; p.BβSer473*) in exon 8 of proband 2. The conservatism analysis revealed that Ala98 and Ser473 presented different conservative states among homologous species. Online bioinformatics software predicted that p.BβAla98Asp and p.BβSer473* were pathogenic. Protein models demonstrated that the p.BβAla98Asp mutation influenced hydrogen bonds between amino acids, and the p.BβSer473* mutation resulted in protein truncation. Conclusion: The dysfibrinogenemia of proband 1 and the hypofibrinogenemia of proband 2 appeared to be related to the p.BβAla98Asp heterozygous missense mutation and the p.BβSer473* heterozygous nonsense mutation, respectively. This is the first ever report of these mutations.
Humans ; Afibrinogenemia/genetics* ; Codon, Nonsense ; Pedigree ; Phenotype ; Fibrinogen/genetics* ; Genotype

OBJECTIVES: To summarize the clinical phenotype and genetic characteristics of children with autosomal dominant mental retardation type 5 caused by SYNGAP1 gene mutations. METHODS: A retrospective analysis was performed on the medical data of 8 children with autosomal dominant mental retardation type 5 caused by SYNGAP1 gene mutations who were diagnosed and treated in the Department of Pediatrics, Xiangya Hospital of Central South University. RESULTS: The mean age of onset was 9 months for the 8 children. All children had moderate-to-severe developmental delay (especially delayed language development), among whom 7 children also had seizures. Among these 8 children, 7 had novel heterozygous mutations (3 with frameshift mutations, 2 with nonsense mutations, and 2 with missense mutations) and 1 had 6p21.3 microdeletion. According to the literature review, there were 48 Chinese children with mental retardation caused by SYNGAP1 gene mutations (including the children in this study), among whom 40 had seizures, and the mean age of onset of seizures was 31.4 months. Frameshift mutations (15/48, 31%) and nonsense mutations (19/48, 40%) were relatively common in these children. In terms of treatment, among the 33 children with a history of epileptic medication, 28 (28/33, 85%) showed response to valproic acid antiepileptic treatment and 16 (16/33, 48%) achieved complete seizure control after valproic acid monotherapy or combined therapy. CONCLUSIONS Children with autosomal dominant mental retardation type 5 caused by SYNGAP1 gene mutations tend to have an early age of onset, and most of them are accompanied by seizures. These children mainly have frameshift and nonsense mutations. Valproic acid is effective for the treatment of seizures in most children.
Child ; Humans ; Intellectual Disability/diagnosis* ; Codon, Nonsense ; Retrospective Studies ; Valproic Acid ; ras GTPase-Activating Proteins/genetics* ; Mutation ; Seizures/genetics*

OBJECTIVES: Hereditary spherocytosis (HS) is the most common hereditary defect of the red cell membrane, mainly characterized by anemia, jaundice, and splenomegaly. Due to the atypical clinical manifestations and negative family history of some patients, as well as the low sensitivity and specificity of traditional laboratory examinations, it is easy for it to escape diagnosis or be misdiagnosed. At present, it has been confirmed that the mutation of ANK1, SPTB, SPTA1, SLC4A1 and EPB42 genes can cause the deletion of their corresponding coding proteins, and thus lead to the defect of erythrocyte membrane. This study aims to analyze the feasibility and clinical application value of HS gene diagnosis. METHODS: Data of 26 patients from Hunan, China with HS admitted to the Department of Hematology, Second Xiangya Hospital of Central South University from January 2018 to September 2021 were retrospectively collected, and their clinical manifestations and results of laboratory examinations were analyzed. Next-generation sequencing (NGS) combined with Sanger sequencing were applied. The mutation of HS pathogenic gene and the variation of uridine diphosphate-glucuronosyl transferase 1 family polypeptide A1 (UGT1A1), a key enzyme in the regulation of bilirubin metabolism, were detected. The results of pathogenic gene variations were interpreted pathogenic gene variations in accordance with the Standards and guidelines for the interpretation of sequence variants published by the American College of Medical Genetics and Genomics (ACMG). The clinical characteristics of patients with different gene variants were analyzed, and the clinical diagnosis and genetic diagnosis were compared. RESULTS: Among the 26 patients with HS, there were 23 cases of anemia, 25 cases of jaundice, 24 cases of splenomegaly, and 14 cases of cholelithiasis. There were 16 cases with family history and 10 cases without family history. The results of HS mutation test were positive in 25 cases and negative in 1 case. A total of 18 heterozygous mutations of HS pathogenic genes were detected in 19 families, among which 14 were pathogenic, 1 was likely pathogenic and 3 were of unknown significance. SPTB mutations (12) and ANK1 mutations (4) were the most common. The main variation types were nonsense mutation (9). There were no significant differences in peripheral blood cell parameters and hemolysis indicators between the SPTB mutant group and the ANK1 mutant group (all P>0.05). The rate of splenectomy in ANK1 mutation group was higher than that in SPTB mutation group, and the difference was statistically significant (χ²=6.970, P=0.014). There were no significant differences in peripheral blood cell parameters and hemolysis indicators among different mutation types (nonsense mutation, frameshift mutation, splice site mutation and missense mutation) (all P>0.05). Among the 18 clinically confirmedpatients, there were 17 cases whose diagnosis is consistent with the genetic diagnosis. Eight patients were clinically suspected, and all of them were confirmed by detection of HS gene mutation. Twenty-four patients with HS underwent UGT1A1 mutation detection, among which 5 patients carried UGT1A1 mutation resulting in a decrease in enzyme activity, and 19 patients had normal enzyme activity. The level of total bilirubin (TBIL) in the group with reduced enzyme activity was higher than that in the group with normal enzyme activity, and the difference was statistically significant (U=22, P=0.038). CONCLUSIONS Most patients with HS have anemia, jaundice and splenomegaly, often accompanied by cholelithiasis. SPTB and ANK1 mutations are the most common mutations in HS pathogenic genes among patients in Hunan, China, and there was no significant correlation between genotype and clinical phenotype. Genetic diagnosis is highly consistent with clinical diagnosis. The decrease of UGT1A1 enzyme activity can lead to the aggravation of jaundice in HS patients. Clinical combined gene diagnosis is beneficial for the rapid and precision diagnosis of HS. The detection of UGT1A1 enzyme activity related gene variation plays an important role in evaluation of HS jaundice.
Humans ; Codon, Nonsense ; Hemolysis ; Retrospective Studies ; Splenomegaly ; Bilirubin

OBJECTIVE: To analyze the clinical characteristics and ZBTB18 gene variant in a child with epilepsy and global developmental delay. METHODS: Clinical data and laboratory examination of the patient were reviewed. Whole exome sequencing (WES) was also carried out for the family trio. RESULTS: The main manifestations of the child included global developmental delay, short stature, epileptic seizures. EEG revealed frequent occurrence of sharp (slow) waves in the right central region during sleeping, with sharp waves occasionally seen in the frontal and right posterior temporal regions. Cranial MRI has shown no obvious abnormality. WES has identified a de novo pathogenic variant in the ZBTB18 gene [NM_205768.3: exon 2: c.1282_1283del (p.Phe428Leufs*72)]. Based on the guidelines from American College of Medical Genetics and Genomics (ACMG), the variant was classified as pathogenic (PS2+PVS1_Moderate+PM2_Supporting). Following treatment with levetiracetam and rehabilitation, the seizures have been controlled for nearly half a year, with improvement of the psychomotor and language development. So far 28 children have been discovered with ZBTB18 gene mutations, and there was a significant difference in the clinical phenotypes of motor retardation, language retardation and epilepsy between those harboring frameshift/nonsense mutations and missense mutations. CONCLUSION The c.1282_1283del (p.Phe428leufs *72) variant of the ZBTB18 probably underlay the autosomal dominant mental disorder type 22 in this child. Compared with missense mutations, frameshift/nonsense mutations may predispose more to motor retardation, delayed language development and epilepsy.
Codon, Nonsense ; Epilepsy/genetics* ; Humans ; Intellectual Disability/genetics* ; Mutation ; Whole Exome Sequencing

OBJECTIVE: To construct a mouse model of Glanzmann's thrombasthenia (GT) with ITGA2B c.2659 C>T (p.Q887X) nonsense mutation by CRISPR/Cas9 technology, and then further explore the expression and function of glycoprotein αIIbβ3 on the surface of platelet membrane. METHODS: The donor oligonucleotide and gRNA vector were designed and synthesized according to the ITGA2B gene sequence. The gRNA and Cas9 mRNA were injected into fertilized eggs with donor oligonucleotide and then sent back to the oviduct of surrogate mouse. Positive F0 mice were confirmed by PCR genotyping and sequence analysis after birth. The F1 generation of heterozygous GT mice were obtained by PCR and sequencing from F0 bred with WT mice, and then homozygous GT mice and WT mice were obtained by mating with each other. The phenotype of the model was then further verified by detecting tail hemorrhage time, saphenous vein bleeding time, platelet aggregation, expression and function of αIIbβ3 on the surface of platelet. RESULTS: The bleeding time of GT mice was significantly longer than that of WT mice (P<0.01). Induced by collagen, thrombin, and adenosine diphosphate (ADP), platelet aggregation in GT mice was significantly inhibited (P<0.01, P<0.01, P<0.05). Flow cytometry analysis showed that the expression of αIIbβ3 on the platelet surface of GT mice decreased significantly compared with WT mice (P<0.01), and binding amounts of activated platelets to fibrinogen were significantly reduced after thrombin stimulation (P<0.01). The spreading area of platelet on fibrinogen in GT mice was significantly smaller than that in WT mice (P<0.05). CONCLUSION A GT mouse model with ITGA2B c.2659 C>T (p.Q887X) nonsense mutation has been established successfully by CRISPR/Cas9 technology. The aggregation function of platelet in this model is defective, which is consistent with GT performance.
Animals ; CRISPR-Cas Systems ; Codon, Nonsense ; Disease Models, Animal ; Fibrinogen/genetics* ; Humans ; Integrin alpha2/genetics* ; Mice ; Oligonucleotides ; Platelet Glycoprotein GPIIb-IIIa Complex/genetics* ; RNA, Guide ; Thrombasthenia/genetics* ; Thrombin/genetics*

OBJECTIVE: To analysis clinical phenotype and potential genetic cause of a family affected with hereditary coagulation factor Ⅻ deficiency. METHODS: The prothrombin time (PT), activated partial thromboplastin time (APTT), fibrinogen (FIB), D-Dimer (D-D), coagulation factor Ⅻ activity (FⅫ:C) and coagulation factor Ⅻ antigen (FⅫ:Ag) were determined for phenotype diagnosis of the proband and his family members(3 generations and 5 people). Targeted capture and whole exome sequencing were performed in peripheral blood sample of the proband. Possible disease-causing mutations of F12 gene were obtained and further confirmed by Sanger sequencing. The corresponding mutation sites of the family members were analyzed afterwards. The online bioinformatics software AutoPVS1 and Mutation Taster was used to predict the effects of mutation sites on protein function. RESULTS: The APTT of the proband was significantly prolonged, reaching 180.9s. FⅫ:C and FⅫ:Ag of the proband was significantly reduced to 0.8% and 4.17%, respectively. The results of whole exome sequencing displayed that there were compound heterozygous mutations in F12 gene of the proband, including the c.1261G＞T heterozygous nonsense mutation in exon 11 (causing p.Glu421*) and the c.251dupG heterozygous frameshift mutation in exon 4 (causing p.Trp85Metfs*53). Both mutations are loss of function mutations with very strong pathogenicity, leading to premature termination of the protein. AutoPVS1 and Mutation Taster software predicted both mutations as pathogenic mutations. The results of Sanger sequencing revealed that c.1261G＞T heterozygous mutation of the proband was inherited from his mother, for which his brother and his daughter were c.1261G＞T heterozygous carriers. Genotype-phenotype cosegregation was observed in this family. CONCLUSION The c.1261G＞T heterozygous nonsense mutation in exon 11 and the c.251dupG heterozygous frameshift mutation in exon 4 of the F12 gene probably account for coagulation factor Ⅻ deficiency in this family. This study reports two novel pathogenic F12 mutations for the first time worldwide.
Blood Coagulation Disorders ; Codon, Nonsense ; Factor XII/genetics* ; Female ; Heterozygote ; Humans ; Male ; Mutation ; Pedigree

OBJECTIVE: To investigate the relationship between the type of FⅧgene mutation and the development of FⅧ inhibitors in patients with severe haemophilia A (HA). METHODS: The medical records of 172 patients with severe hemophilia A from January 2009 to September 2020 were reviewed. The types of FⅧgene mutations and the production of factor Ⅷ inhibitors were collected and divided into high-risk mutation group ( intron 1 inversions, large deletions, nonsense mutations), low-risk mutation group (missense mutations, small deletions and insertions, splice site mutations) and intron 22 inversions group. The correlation of FⅧgenotype and the production of FⅧ inhibitors in patients with HA were analyzed. RESULTS: Among 172 patients with severe HA, 21 cases(12.21%) developed FⅧ inhibitors. The cumulative incidence of FⅧ inhibitor development was 32%(10/31) in high risk group (75% patients with large deletions, 43% patients with intron 1 inversions, 20% patients with nonsense mutations) and 5%(2/43) in low risk group(6% patients with missense mutations, 5% patients with small deletions or insertions and 0% patient with a splice site mutation) and 9%(9/98) in intron 22 inversions group. Compared with the risk of FⅧ inhibitor development in intron 22 inversions group, the risk of FⅧ inhibitor development in high risk group was higher (OR＝4.7, 95% CI： 1.7-13.0), the risk of FⅧ inhibitor development in low risk group was equal (OR＝0.5, 95% CI： 0.1-2.3). Compared with the risk of inhibitor development in low risk group, the risk of FⅧ inhibitor development in high risk group was higher (OR＝9.8, 95% CI： 2.0-48.7). CONCLUSION Gene mutations of patients with severe HA in high-risk group which include intron 1 inversions, large deletions, nonsense mutations are a risk factor for FⅧ inhibitor production.
Codon, Nonsense ; DNA Mutational Analysis ; Factor VIII/genetics* ; Hemophilia A/genetics* ; Humans ; Introns ; Mutation