Objective To investigate the potential value of crocetin(CRO)in the treatment of diabetic retinopathy(DR)and its effect on signal transducer and activator of transcription 3(STAT3)activity.Methods(1)In vitro experi-ment:RPE cells were divided into C group,HG group,HG+5/10/20CRO groups and HG+20CRO+Colivelin TFA(C-TFA)group.Cells in the C group were cultured with normal glucose medium(5 mmol·L-1).Cells in the HG+5/10/20CRO groups were cultured in high glucose medium(25 mmol·L-1)with 5,10 and 20 μmol·L-1 CRO added.Cells in the HG+20CRO+C-TFA group were cultured in high glucose medium with 20 μmol·L-1 CRO and 0.5 μmol·L-1 C-TFA added.The cells in each group were cultured for 48 h.Cell viability,proliferation and apoptosis were detected using MTT,EdU staining,and TUNEL,respectively.Meanwhile,the levels of oxidative stress and inflammatory factors were detected by ELISA,and the activation level of STAT3 was detected by Western blot.(2)In vivo experiment:Rats were divided into 6 groups:NC group,DR group,20/40/80CRO groups and 80CRO+C-TFA group.NC group was normal control rats,and the other groups were STZ-induced DR model rats.NC group and DR group were gavaged with 5 g·L-1 CMC-Na.20/40/80CRO groups were gavaged with 20,40 and 80 mg·kg-1 of CRO.80CRO+C-TFA group was gavaged with 80 mg·kg-1 of CRO,and 1 mg·kg-1 of C-TFA was injected intraperitoneally.The treatment cycle was 4 weeks.HE staining and TUNEL staining were employed to examine retinal morphology and apoptosis.The concentrations of oxidative stress markers and inflammatory factors in the retina were measured using ELISA,while the activation level of STAT3 in the retina was as-sessed through Western blot analysis.Results(1)In vitro experiments:Compared with the HG group,the HG+5/10/20CRO groups exhibited increased relative cell viability,SOD and GPx activities,and EdU+cell ratio,but decreased TUNEL+cell ratio,MDA content,TNF-α,IL-1β and IL-6 levels,and STAT3 phosphorylation level(P＜0.05).Compared with the HG+20CRO group,the abovementioned indicators of RPE cells in the HG+20CRO+C-TFA group were reversed(all P＜0.05).(2)In vivo experiments:Compared with the DR group,the 20/40/80CRO groups exhibited increased SOD and GPx activities,but decreased proportion of retinal TUNEL+cells,retinal MDA content,TNF-α,IL-1β and IL-6 levels,and retinal STAT3 phosphorylation level(all P＜0.05).Compared with the 80CRO group,the abovementioned indicators of reti-nal cell in the 80CRO+C-TFA group were reversed(all P＜0.05).Conclusion CRO can alleviate high glucose-induced rat RPE cell injury and retinal injury of DR rats by inhibiting STAT3 activity.

Objective:To explore the clinical and genetic characteristics of X-linked intellectual disability associated with HUWE1 gene variants.Methods:A cases series study retrospectively analyzed the clinical data of 6 children with HUWE1 gene variants. The children were identified from the Third Affiliated Hospital of Zhengzhou University, the First Affiliated Hospital of Zhengzhou University, the First Affiliated Hospital of Henan University of Chinese Medicine, and Guangzhou Women and Children′s Medical Center of Guangzhou Medical University between April 2021 and July 2023.The data included sex, age, dysmorphic features, intellectual and motor development, seizure history, neuroimaging findings, family history, and genetic results was analyzed.Results:A total of 6 children, including 5 boys and 1 girl. The age of onset ranged from 1 day to 3 years. All children presented with varying degrees of intellectual disability, with or without motor developmental delay. Dysmorphic features were observed in 4 children, including microcephaly in 3 children. Short stature were observed in 3 children. One child was diagnosed with autism spectrum disorders and 1 child had seizures. Two boys had relevant maternal family histories of febrile seizures and mild intellectual disability, respectively. Abnormal neuroimaging findings were presented in 4 children, including cerebral dysplasia (1 child), prominent supratentorial ventricles (1 child), and mild white matter demyelination (2 children). Whole-exome sequencing identified 5 missense variants and 1 in-frame deletion variant. Five variants were novel and previously unreported (c.12290C>T, c.12701T>C, c.9875C>T, c.9641A>T and c.10313_10315del). The variants in 4 boys were maternally inherited, while the remaining 2 children had de novo variants. The child with the in-frame deletion variant (c.10313_10315del) presented with the most severe phenotype, exhibiting symptoms from 1 day of age, absent cognitive development, feeding difficulties, and congenital laryngeal chondrodysplasia. He was lost to follow-up at 3 months of age after treatment was withdrawn. The age at the last follow-up for the remaining 5 children ranged from 2 years and 10 months to 17 years. A boy with seizures died at 2 years and 10 months of age. The remaining 4 children were able to walk independently at the last follow-up, although their developmental progress was slow. Conclusions:HUWE1 gene related X-linked intellectual disability is characterized by varying degrees of developmental delay and intellectual disability, frequently accompanied by microcephaly, short stature, and occasionally by seizures and autism spectrum disorders. Missense variants are more common and the in-frame deletion variant appears to be associated with a particularly severe phenotypic presentation.

Objective To investigate the potential value of crocetin(CRO)in the treatment of diabetic retinopathy(DR)and its effect on signal transducer and activator of transcription 3(STAT3)activity.Methods(1)In vitro experi-ment:RPE cells were divided into C group,HG group,HG+5/10/20CRO groups and HG+20CRO+Colivelin TFA(C-TFA)group.Cells in the C group were cultured with normal glucose medium(5 mmol·L-1).Cells in the HG+5/10/20CRO groups were cultured in high glucose medium(25 mmol·L-1)with 5,10 and 20 μmol·L-1 CRO added.Cells in the HG+20CRO+C-TFA group were cultured in high glucose medium with 20 μmol·L-1 CRO and 0.5 μmol·L-1 C-TFA added.The cells in each group were cultured for 48 h.Cell viability,proliferation and apoptosis were detected using MTT,EdU staining,and TUNEL,respectively.Meanwhile,the levels of oxidative stress and inflammatory factors were detected by ELISA,and the activation level of STAT3 was detected by Western blot.(2)In vivo experiment:Rats were divided into 6 groups:NC group,DR group,20/40/80CRO groups and 80CRO+C-TFA group.NC group was normal control rats,and the other groups were STZ-induced DR model rats.NC group and DR group were gavaged with 5 g·L-1 CMC-Na.20/40/80CRO groups were gavaged with 20,40 and 80 mg·kg-1 of CRO.80CRO+C-TFA group was gavaged with 80 mg·kg-1 of CRO,and 1 mg·kg-1 of C-TFA was injected intraperitoneally.The treatment cycle was 4 weeks.HE staining and TUNEL staining were employed to examine retinal morphology and apoptosis.The concentrations of oxidative stress markers and inflammatory factors in the retina were measured using ELISA,while the activation level of STAT3 in the retina was as-sessed through Western blot analysis.Results(1)In vitro experiments:Compared with the HG group,the HG+5/10/20CRO groups exhibited increased relative cell viability,SOD and GPx activities,and EdU+cell ratio,but decreased TUNEL+cell ratio,MDA content,TNF-α,IL-1β and IL-6 levels,and STAT3 phosphorylation level(P＜0.05).Compared with the HG+20CRO group,the abovementioned indicators of RPE cells in the HG+20CRO+C-TFA group were reversed(all P＜0.05).(2)In vivo experiments:Compared with the DR group,the 20/40/80CRO groups exhibited increased SOD and GPx activities,but decreased proportion of retinal TUNEL+cells,retinal MDA content,TNF-α,IL-1β and IL-6 levels,and retinal STAT3 phosphorylation level(all P＜0.05).Compared with the 80CRO group,the abovementioned indicators of reti-nal cell in the 80CRO+C-TFA group were reversed(all P＜0.05).Conclusion CRO can alleviate high glucose-induced rat RPE cell injury and retinal injury of DR rats by inhibiting STAT3 activity.

Objective:To explore the clinical and genetic characteristics of X-linked intellectual disability associated with HUWE1 gene variants.Methods:A cases series study retrospectively analyzed the clinical data of 6 children with HUWE1 gene variants. The children were identified from the Third Affiliated Hospital of Zhengzhou University, the First Affiliated Hospital of Zhengzhou University, the First Affiliated Hospital of Henan University of Chinese Medicine, and Guangzhou Women and Children′s Medical Center of Guangzhou Medical University between April 2021 and July 2023.The data included sex, age, dysmorphic features, intellectual and motor development, seizure history, neuroimaging findings, family history, and genetic results was analyzed.Results:A total of 6 children, including 5 boys and 1 girl. The age of onset ranged from 1 day to 3 years. All children presented with varying degrees of intellectual disability, with or without motor developmental delay. Dysmorphic features were observed in 4 children, including microcephaly in 3 children. Short stature were observed in 3 children. One child was diagnosed with autism spectrum disorders and 1 child had seizures. Two boys had relevant maternal family histories of febrile seizures and mild intellectual disability, respectively. Abnormal neuroimaging findings were presented in 4 children, including cerebral dysplasia (1 child), prominent supratentorial ventricles (1 child), and mild white matter demyelination (2 children). Whole-exome sequencing identified 5 missense variants and 1 in-frame deletion variant. Five variants were novel and previously unreported (c.12290C>T, c.12701T>C, c.9875C>T, c.9641A>T and c.10313_10315del). The variants in 4 boys were maternally inherited, while the remaining 2 children had de novo variants. The child with the in-frame deletion variant (c.10313_10315del) presented with the most severe phenotype, exhibiting symptoms from 1 day of age, absent cognitive development, feeding difficulties, and congenital laryngeal chondrodysplasia. He was lost to follow-up at 3 months of age after treatment was withdrawn. The age at the last follow-up for the remaining 5 children ranged from 2 years and 10 months to 17 years. A boy with seizures died at 2 years and 10 months of age. The remaining 4 children were able to walk independently at the last follow-up, although their developmental progress was slow. Conclusions:HUWE1 gene related X-linked intellectual disability is characterized by varying degrees of developmental delay and intellectual disability, frequently accompanied by microcephaly, short stature, and occasionally by seizures and autism spectrum disorders. Missense variants are more common and the in-frame deletion variant appears to be associated with a particularly severe phenotypic presentation.

Objective:To investigate the DMD genetic variants of the Chinese population with Duchenne (DMD) and Becker muscular dystrophies (BMD).Methods:A cross-sectional study was conducted on 2 690 unrelated patients with DMD and BMD aged 0-18 who visited the Genetic and Prenatal Diagnosis Center of the First Affiliated Hospital of Zhengzhou University from January 2005 to February 2022. The clinical data, such as gender, age, clinical manifestations, and address, were collected. Multiplex ligation-dependent probe amplification, next generation sequencing panel, Sanger sequencing, and PCR amplification were used to detect the variants of the DMD gene in the patients, whose clinical information and gene detection results were descriptively analyzed.Results:The 2 690 patients included 2 648 males and 42 females, with an age of 6.0 (4.0, 9.0) years. The serum creatine kinase increased in all patients. Pathogenic DMD gene variants were detected in the 2 618 patients, including 1 875 cases (71.6%) large deletions, 231 cases (8.8%) duplications, and 512 cases (19.6%) small variants. Among the deletion variants, the deletion of 3 exons was the most common, accounting for 15.4% (288/1 875); and hotspot deletion involved exons 45 to 50, accounting for 6.3% (119/1 875). Exon 2 was the most common type duplication region, accounting for 13.0% (30/231). Small variants were distributed in all 79 exons of the DMD gene, with no hotspots. In addition, the 46 small variants were previously unreported.Conclusion:Exon deletion is the most common type of DMD gene variant, followed by small variants and exon duplication.

Objective:To explore the significance and interpretation of low-depth whole-genome copy number variation sequencing (CNV-seq) in prenatal diagnosis in detecting DMD gene variations in fetuses without a family history of genetic diseases, and to investigate the results of family testing. Methods:Retrospectively collected case data of 16 fetuses with DMD gene deletions or duplications detected by low-depth whole-genome CNV-seq from December 2019 to August 2023 at the First Affiliated Hospital of Zhengzhou University. Amniotic fluid or chorionic villus samples and peripheral blood from family members were collected for all 16 cases, and genomic DNA was extracted. The fetal chromosomal copy number variations were detected using CNV-seq technology and the DMD gene deletions or duplications were verified by multiplex ligation-dependent probe amplification (MLPA), followed by family validation to trace the source of variation. The pathogenicity of the DMD gene deletion or duplication fragments was analyzed based on online Mendelian genetics databases and family validation results. Results:All 16 cases denied a family history of monogenic diseases. The indications for CNV-seq prenatal diagnosis were high-risk Down syndrome screening in nine cases, advanced maternal age in two cases, abnormal fetal ultrasound in three cases, and non-invasive prenatal DNA testing suggesting X chromosome abnormalities in two cases. CNV-seq results indicated nine cases of DMD gene duplication variations and seven cases of DMD gene deletion variations. MLPA validation confirmed results consistent with CNV-seq detection. Family analysis showed that three cases were de novo variations, 12 cases were inherited from the mother, one case had a mother with normal peripheral blood testing but a sister carrying the same variation, suggesting a high possibility of the mother being a carrier of gonadal mosaic. The likelihood of pathogenic variation was high in seven cases of deletion; nine cases were duplication variations, four of which were located within the DMD gene and could potentially disrupt the gene, leading to disease, while the other five variations were located in the 5' untranslated region or 3' untranslated region, considered benign variations. Conclusions:Low-depth whole-genome CNV-seq can effectively detect large deletion and duplication variations of the DMD gene in fetuses without a family history, preventing the birth of children with de novo variations. However, the pathogenicity of fetuses with large DMD gene duplications should be assessed based on family validation. When the duplication region includes the 5' untranslated region or 3' untranslated region of the DMD gene, it is more likely to be a polymorphic variation.

OBJECTIVE: To explore the genetic basis for a Chinese patient suspected for Canavan disease. METHODS: Whole exome sequencing (WES) was carried out for the proband, and candidate variants were verified by Sanger sequencing of the proband, her parents and brother. Prenatal diagnosis was provided to her mother by chorionic villi sampling (CVS) upon her subsequent pregnancy. RESULTS: The proband, a 4-month-old female infant, had manifested drowsiness, hypotonia and apathy. Urine metabolism screening showed elevated N-acetylaspartic acid. Cranial magnetic resonance imaging revealed abnormal myelination and multiple abnormal signals in large brain areas. WES revealed that the proband has harbored compound heterozygous variants of the ASPA gene, namely c.187A>G (p.Arg63Gly) in exon 1 and c.634+1G>A (P.?) in exon 4. Sanger sequencing confirmed that the c.187A>G (p.Arg63Gly) and c.634+1G>A (p.?) variants were respectively inherited from her mother and father. Her phenotypically normal brother has carried a heterozygous c.634+1G>A (p.?) variant. Prenatal diagnosis by CVS indicated that the fetus was a heterozygous carrier of the c.187A>G variant. CONCLUSION WES can facilitate the diagnosis of Canavan disease, particularly for those lacking specific phenotypes of the disease. The compound heterozygous variants of the ASPA gene probably underlay the Canavan disease in this patient, and the result has enabled prenatal diagnosis for this family.
Canavan Disease/genetics* ; China ; Female ; Humans ; Male ; Mutation ; Pedigree ; Pregnancy ; Prenatal Diagnosis

OBJECTIVE: To explore the genetic etiology of a patient with mucopolysaccharidosis type II (MPSII). METHODS: The IDS gene of the proband and his mother was detected by Sanger sequencing, agarose gel electrophoresis, real-time PCR and multiple ligation-dependent probe amplification (MLPA). Prenatal diagnosis was performed on amniotic fluid sample. RESULTS: Agarose gel electrophoresis, real-time PCR, and MLPA all showed that exon 2 of IDS gene of the proband was deleted, for which his mother was normal. Prenatal diagnosis showed that the fetus was a normal male. CONCLUSION The de novo deletion of exon 2 of the IDS gene probably underlay the MPSII in this patient. Above finding has broadened the mutation spectrum of the IDS gene. The combined methods for the detection of IDS gene mutations could make accurate prenatal diagnosis for MPSII.
China ; Exons ; Female ; Humans ; Male ; Mucopolysaccharidosis II/genetics* ; Mutation ; Pedigree ; Pregnancy ; Prenatal Diagnosis/methods*

OBJECTIVE: To assess the value of re-sampling for patients who had failed non-invasive prenatal testing (NIPT) due to low cell-free fetal DNA (cffDNA) fraction. METHODS: Clinical data of 20 387 patients undergoing NIPT test was reviewed. The patients were re-sampled when initial blood test did not yield a result due to cffDNA fraction. The results were analyzed, and the outcome of pregnancy was followed up. RESULTS: Among all samples, 17 (0.08%) had failed to yield a result due to low cffDNA fraction, all of which accepted re-sampling. A result was attained in 16 cases, with a success rate of 94.12%. Only one sample had failed the re-test. CONCLUSION For patients who had failed the initial NIPT due to low cffDNA fraction, re-sampling should be considered with gestational week and ultrasound results taken into consideration.
Aneuploidy ; Cell-Free Nucleic Acids/genetics* ; DNA/genetics* ; Female ; Fetus ; Humans ; Pregnancy ; Prenatal Diagnosis

Objective:To analyze the clinical manifestations of congenital cataract in 12 families and gene variants causing the disease.Methods:The method of pedigree investigation was adopted.Clinical data of 27 patients from 12 Chinese Han families with congenital cataract were collected, and genomic DNA was extracted from peripheral blood samples of patients and family members.Candidate variants were screened by next generation sequencing and were verified by Sanger sequencing.Population frequency of the variants were obtained through the Genome Arrgregation Database (gnomAD).Pathogenicity of variants was analyzed through the Human Gene Mutation Database (HGMD), Database of Single Nucleotide Polymorphism (dbSNP) and PubMed, and the mutation effect was interpreted by protein prediction softwares including SIFT, PolyPhen_2 and MutationTaster.The conservation analysis of amino acid sequences of variants was performed by GERP+ + software.Diagnosis was confirmed by clinical ophthalmic phenotype, medical history and mutation analysis.This study adhered to the Declaration of Helsinki.The study protocol was approved by the Ethics Committee of the First Affiliated Hospital of Zhengzhou University (No.KS-2018-KY-36).Written informed consent was obtained from all subjects and their guardians.Results:Pathogenic genetic variants were found in all the 12 families, 9 of which had known pathogenic variants including MIP c.97C>T, GJA8 c.593G>A, CRYBA4 c.277T>C, CRYBB2 c.563G>A and c.436G>C, CRYGC c.470G>A, CRYGD c.70C>A, PAX2 c.70dupG as well as OCRL E5-E16dup, and 3 novel potential pathogenic variants including CRYGD c.422delG, ELP4 c.886C>A and CRYBB2 c.434G>C. CRYGD c.422delG could lead to the early termination of translation of protein products, which was pathogenic.The nucleotide and amino acid sites of ELP4 c.886C>A and CRYBB2 c.434G>C were highly conserved among species, and were predicted as harmful.The 12 families were consistent with co-segregation. Conclusions:CRYGD c.422delG, ELP4 c.886C>A and CRYBB2 c.434G>C may be novel pathogenic variants of congenital cataract.