Three-way contact analysis characterizes the higher order organization of the Tcra locus.

The generation of highly diverse antigen receptors in T and B lymphocytes relies on V(D)J recombination. The enhancer Eα has been implicated in regulating the accessibility of Vα and Jα genes through long-range interactions during rearrangements of the T-cell antigen receptor gene Tcra. However, direct evidence for Eα physically mediating the interaction of Vα and Jα genes is still lacking. In this study, we utilized the 3C-HTGTS assay, a chromatin interaction technique based on 3C, to analyze the higher order chromatin structure of the Tcra locus. Our analysis revealed the presence of sufficient information in the 3C-HTGTS data to detect multiway contacts. Three-way contact analysis of the Tcra locus demonstrated the co-occurrence of the proximal Jα genes, Vα genes and Eα in CD4+CD8+ double-positive thymocytes. Notably, the INT2-TEAp loop emerged as a prominent structure likely to be responsible for bringing the proximal Jα genes and the Vα genes into proximity. Moreover, the enhancer Eα utilizes this loop to establish physical proximity with the proximal Vα gene region. This study provides insights into the higher order chromatin structure of the Tcra locus, shedding light on the spatial organization of chromatin and its impact on V(D)J recombination.

[Clinical features and genetic analysis of 17 Chinese pedigrees affected with X-linked intellectual disability].

OBJECTIVE: To analyze the clinical features and genetic etiology of 17 Chinese pedigrees affected with X-linked intellectual disability (XLID). METHODS: Seventeen pedigrees affected with unexplained intellectual disability which had presented at Henan Provincial People's Hospital from May 2021 to May 2023 were selected as the study subjects. Clinical data of the probands and their pedigree members were collected. Trio-whole exome sequencing (Trio-WES), Sanger sequencing and X chromosome inactivation (XCI) analysis were carried out. Pathogenicity of candidate variants was predicted based on the guidelines from the American College of Medical Genetics and Genomics and co-segregation analysis. RESULTS: The 17 probands, including 9 males and 8 females with an age ranging from 0.6 to 8 years old, had all shown mental retardation and developmental delay. Fourteen variants were detected by genetic testing, which included 4 pathogenic variants (MECP2: c.502C>T, MECP2: c.916C>T/c.806delG, IQSEC2: c.1417G>T), 4 likely pathogenic variants (MECP2: c.1157_1197del/c.925C>T, KDM5C: c.2128A>T, SLC6A8: c.1631C>T) and 6 variants of uncertain significance (KLHL15: c.26G>C, PAK3: c.970A>G/c.1520G>A, GRIA3: c.2153C>G, TAF1: c.2233T>G, HUWE1: c.10301T>A). The PAK3: c.970A>G, GRIA3: c.2153C>G and TAF1: c.2233T>G variants were considered as the genetic etiology for pedigrees 12, 14 and 15 by co-segregation analysis, respectively. The proband of pedigree 13 was found to have non-random XCI (81:19). Therefore, the PAK3: c.1520G>A variant may underlie its pathogenesis. CONCLUSION: Trio-WES has attained genetic diagnosis for the 17 XLID pedigrees. Sanger sequencing and XCI assay can provide auxiliary tests for the diagnosis of XLID.

Risk factors and healing factors for pharyngocutaneous fistula after total laryngectomy for laryngeal cancer: An epidemiological study.

To analyse the risk factors and healing factors of pharyngocutaneous fistula (PCF) in patients with laryngeal cancer after total laryngectomy, and to explore the relevant epidemiology. A retrospective analysis was conducted on laryngeal cancer patients who underwent total laryngectomy in our hospital from January 2010 to December 2022. The 349 patients included in the study were divided into a PCF group of 79 and a non-PCF group of 270. Perform one-way analysis of variance and multivariate logistic analysis on various data of patients included in the statistics, and analyse the risk factors and healing factors of PCF. Smoking, history of radiation therapy for laryngeal cancer, history of chemotherapy for laryngeal cancer, tumour location (larynx, pharynx, oesophagus), preoperative albumin, postoperative proteinaemia, <99 haemoglobin, postoperative haemoglobin, postoperative C-reactive protein (CRP) level are the risk factors for PCF. Also, radiation therapy and postoperative proteinaemia were the main reasons for preventing PCF healing. Smoking history, laryngeal cancer, radiation therapy, albumin, haemoglobin and CRP are risk factors for postoperative PCF after total laryngectomy, while radiation therapy and postoperative hypoalbuminaemia are key factors affecting PCF healing.

Screening and identification of miR-181a-5p in oral squamous cell carcinoma and functional verification in vivo and in vitro.

BACKGROUND: Oral squamous cell carcinoma (OSCC) is a common malignant tumor associated with poor prognosis. MicroRNAs (miRNAs) play crucial regulatory roles in the cancer development. However, the role of miRNAs in OSCC development and progression is not well understood. METHODS: We sought to establish a dynamic Chinese hamster OSCC animal model, construct miRNA differential expression profiles of its occurrence and development, predict its targets, and perform functional analysis and validation in vitro. RESULTS: Using expression and functional analyses, the key candidate miRNA (miR-181a-5p) was selected for further functional research, and the expression of miR-181a-5p in OSCC tissues and cell lines was detected. Subsequently, transfection technology and a nude mouse tumorigenic model were used to explore potential molecular mechanisms. miR-181a-5p was significantly downregulated in human OSCC specimens and cell lines, and decreased miR-181a-5p expression was observed in multiple stages of the Chinese hamster OSCC animal model. Moreover, upregulated miR-181a-5p significantly inhibited OSCC cell proliferation, colony formation, invasion, and migration; blocked the cell cycle; and promoted apoptosis. BCL2 was identified as a target of miR-181a-5p. BCL2 may interact with apoptosis- (BAX), invasion- and migration- (TIMP1, MMP2, and MMP9), and cell cycle-related genes (KI67, E2F1, CYCLIND1, and CDK6) to further regulate biological behavior. Tumor xenograft analysis indicated that tumor growth was significantly inhibited in the high miR-181a-5p expression group. CONCLUSION: Our findings indicate that miR-181a-5p can be used as a potential biomarker and provide a novel animal model for mechanistic research on oral cancer.

A role of the CTCF binding site at enhancer Eα in the dynamic chromatin organization of the Tcra-Tcrd locus.

The regulation of T cell receptor Tcra gene rearrangement has been extensively studied. The enhancer Eα plays an essential role in Tcra rearrangement by establishing a recombination centre in the Jα array and a chromatin hub for interactions between Vα and Jα genes. But the mechanism of the Eα and its downstream CTCF binding site (here named EACBE) in dynamic chromatin regulation is unknown. The Hi-C data showed that the EACBE is located at the sub-TAD boundary which separates the Tcra-Tcrd locus and the downstream region including the Dad1 gene. The EACBE is required for long-distance regulation of the Eα on the proximal Vα genes, and its deletion impaired the Tcra rearrangement. We also noticed that the EACBE and Eα regulate the genes in the downstream sub-TAD via asymmetric chromatin extrusion. This study provides a new insight into the role of CTCF binding sites at TAD boundaries in gene regulation.

The role of chromatin organizer Satb1 in shaping TCR repertoire in adult thymus.

T cells recognize the universe of foreign antigens with a diverse repertoire of T cell receptors generated by V(D)J recombination. Special AT-rich binding protein 1 (Satb1) is a chromatin organizer that plays an essential role in T cell development. Previous study has shown that Satb1 regulates the re-induction of recombinase Rag1 and Rag2 in CD4+CD8+ thymocytes, affecting the secondary rearrangement of the Tcra gene. Here, we detected the repertoires of four TCR genes, Tcrd, Tcrg, Tcrb, and Tcra, in the adult thymus, and explored the role of the Satb1 in shaping the TCR repertoires. We observed a strong bias in the V and J gene usages of the Tcrd and Tcrg repertoires in WT and Satb1-deleted thymocytes. Satb1 deletion had few effects on the V(D)J rearrangement and repertoire of the Tcrg, Tcrd, and Tcrb genes. The Tcra repertoire was severely impaired in Satb1-deleted thymocytes, while the primary rearrangement was relatively normal. We also found the CDR3 length of TCRα chain was significantly longer in Satb1-deleted thymocytes, which can be explained by the strong bias of the proximal Jα usage. Our results showed that Satb1 plays an essential role in shaping TCR repertoires in αß T cells.

[Identification of SPAST gene variant in a pedigree affected with hereditary spastic paraplegia type 4].

OBJECTIVE: To explore the genetic basis for a pedigree affected with hereditary spastic paraplegia type 4 (HSP4). METHODS: Peripheral venous blood samples were taken from members of the four-generation pedigree and 50 healthy controls for the extraction of genomic DNA. Genes associated with peripheral neuropathy and hereditary spastic paraplegia were captured and subjected to targeted capture and next-generation sequencing. The results were confirmed by Sanger sequencing. RESULTS: DNA sequencing suggested that the proband has carried a heterozygous c.1196C>G variant in exon 9 of the SPAST gene, which can cause substitution of serine by threonine at position 399 (p.Ser399Trp) and lead to change in the protein function. The same variant was also detected in other patients from the pedigree but not among unaffected individuals or the 50 healthy controls. Based on the ACMG 2015 guidelines, the variant was predicted to be possibly pathogenic. CONCLUSION: The c.1196C>G variant of the SPAST gene probably underlay the HSP4 in this pedigree.

Long noncoding RNA SOX2-OT facilitates laryngeal squamous cell carcinoma development by epigenetically inhibiting PTEN via methyltransferase EZH2.

Long noncoding RNAs (lncRNAs) play important roles in the initiation and progression of various cancers, including laryngeal squamous cell carcinoma (LSCC). Recently, lncRNA Sox2 overlapping transcript (SOX2-OT) has been identified as an oncogene in various cancers. However, the functional role and the regulatory mechanism of SOX2-OT in LSCC remains unclear. In this study, we found that SOX2-OT expression was increased and negatively correlated with PTEN expression in LSCC tissues. Furthermore, SOX2-OT overexpression promoted LSCC cell proliferation, migration, invasion, and suppressed cell apoptosis in vitro, as well as facilitated the in vivo tumorigenicity. By contrast, SOX2-OT silencing exerted the opposite effect. Mechanically, SOX2-OT interacted with EZH2 and recruited EZH2 to induce H3K27me3 and epigenetically inhibited PTEN expression in LSCC cells. Additionally, EZH2 silencing and PTEN overexpression significantly abrogated the SOX2-OT overexpression-mediated promotion of LSCC cell malignant behavior. Collectively, our findings demonstrate that SOX2-OT inhibits PTEN expression to facilitate LSCC development through EZH2-mediated H3K27me3. © 2019 IUBMB Life, 71(9):1230-1239, 2019.

[Identification of two novel Parkin gene mutations in a patient affected with Juvenile Parkinson's syndrome].

OBJECTIVE: To explore the clinical and genetic features of a patient suspected with Juvenile Parkinson's syndrome (JP). METHODS: Clinical features of the patient were analyzed. Genomic DNA of the patient and his parents was extracted from peripheral blood samples and sequenced by exome capture sequencing. The nature and impact of detected mutations were predicted and validated. RESULTS: The patient displayed typical features including resting tremor, bradykinesia, rigidity, but with excellent response to low dose levodopa. DNA sequencing showed that she has carried compound heterozygous mutations of the Parkin gene, namely c.1381dupC and c.619-1G>C, which were respectively inherited from his mother and father. Neither mutation was reported previously. Bioinformatic analysis predicted that both mutations are pathogenic. CONCLUSION: The patient has JP caused by mutations of the Parkin gene. Exome capture sequencing is an accurate and efficient method for genetic diagnosis of such disease.

[Analysis of MYO7A gene mutation in a family with non-syndromic autosomal recessive deafness].

OBJECTIVE: To explore the genetic basis for a family with non-syndromic autosomal recessive deafness. METHODS: The proband and her parents were subjected to physical and audiological examinations. With genomic DNA extracted from peripheral blood samples, next-generation sequencing was carried out using a panel for deafness genes. Suspected mutation was validated by Sanger sequencing and qPCR analysis of her parents. RESULTS: The proband presented bilateral severe sensorineural hearing loss at three days after birth. Her auditory threshold was 110-120 dBnHL but with absence of vestibular and retinal symptoms. Her brother also had deafness but her parents were normal. No abnormality was found upon physical examination of her family members, while audiological examination showed no middle ear or retrocochlear diseases. Next-generation sequencing identified compound heterozygous mutations of the MYO7A gene, including a previously known c.462C>A (p. Cys154Ter) and a novel EX43_46 Del, which were respectively derived from her mother and father. CONCLUSION: The compound heterozygous mutations of the MYO7A gene probably underlie the disease in this family. Our findings has enriched the mutation spectrum for non-syndromic autosomal recessive deafness 2.

[Prenatal diagnosis of a fetus affected with Finnish type congenital nephrotic syndrome].

OBJECTIVE: To explore the genetic basis for a fetus suspected for congenital nephrotic syndrome of Finland (CNF). METHODS: Genomic DNA was extracted from peripheral and umbilical cord blood samples derived from both parents and the fetus. Potential variants were detected by using next-generation sequencing. Suspected variants were confirmed by Sanger sequencing. RESULTS: The fetus was found to carry compound heterozygous variants c.1440+1G>A and c.925G>T of the NPHS1 gene, which were respectively inherited from its mother and father. CONCLUSION: Identification of the compound heterozygous NPHS1 variants has enabled diagnosis of CNF in the fetus and genetic counseling for the affected family.

A dominant variant in the PDE1C gene is associated with nonsyndromic hearing loss.

Identification of genes with variants causing non-syndromic hearing loss (NSHL) is challenging due to genetic heterogeneity. The difficulty is compounded by technical limitations that in the past prevented comprehensive gene identification. Recent advances in technology, using targeted capture and next-generation sequencing (NGS), is changing the face of gene identification and making it possible to rapidly and cost-effectively sequence the whole human exome. Here, we characterize a five-generation Chinese family with progressive, postlingual autosomal dominant nonsyndromic hearing loss (ADNSHL). By combining population-specific mutation arrays, targeted deafness genes panel, whole exome sequencing (WES), we identified PDE1C (Phosphodiesterase 1C) c.958G>T (p.A320S) as the disease-associated variant. Structural modeling insights into p.A320S strongly suggest that the sequence alteration will likely affect the substrate-binding pocket of PDE1C. By whole-mount immunofluorescence on postnatal day 3 mouse cochlea, we show its expression in outer (OHC) and inner (IHC) hair cells cytosol co-localizing with Lamp-1 in lysosomes. Furthermore, we provide evidence that the variant alters the PDE1C hydrolytic activity for both cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP). Collectively, our findings indicate that the c.958G>T variant in PDE1C may disrupt the cross talk between cGMP-signaling and cAMP pathways in Ca2+ homeostasis.

[Analysis of pathological mutation in a Chinese pedigree affected with familial exudative vitreoretinopathy].

OBJECTIVE: To detect potential mutation in a Chinese pedigree affected with familial exudative vitreoretinopathy (FEVR). METHODS: Clinical data of the pedigree was collected. Coding regions of candidate genes were amplified by PCR and subjected to next generation sequencing (NGS). Suspected mutations were verified by Sanger sequencing and segregation analysis. RESULTS: Two novel heterozygous mutations (c.1695dupC and c.552-563del) were respectively detected in the LRP5 and ZNF408 genes in the proband. Both mutations were inherited from the affected mother. By Sanger sequencing, the c.552-563del mutation was also detected among unaffected members, while the c.1695dupC mutation was only detected in affected members from the pedigree and was not recorded by the HGMD, NCBI, or 1000 genome database. Upon prenatal diagnosis, the fetus was found to carry the same mutations. CONCLUSION: Combined NGS and Sanger sequencing not only can reduce the time required for diagnosis but also enable accurate prenatal diagnosis for FEVR.

[Identification of a novel EXT1 mutation in a pedigree affected with hereditary multiple exostosis].

OBJECTIVE To detect potential mutations of the EXT1 and EXT2 genes in a pedigree affected with hereditary multiple exostosis (HME). METHODS For a four-generation family with 7 affected individuals from 17 family members,genomic DNA was extracted from peripheral venous blood samples. All exons of the EXT1 and EXT2 genes were screened for potential mutation by PCR and Sanger sequencing. RESULTS A novel heterozygous frameshift mutation c.1202delT (p.I401Tfs*2)was found in exon 4 of the EXT1 gene in the proband and the other 6 affected individuals. The same mutation was not detected among the healthy members from the family. The mutation has given rise a truncated EXT1 protein with loss of 345 amino acids. CONCLUSION A novel frameshift mutation of the EXT1 gene has been identified in a pedigree affected with HME, which has enriched the mutational spectrum of the EXT1 gene and may facilitate genetic counseling and prenatal diagnosis for the family.

[Phenotypic and genetic analysis of a child carrying a 17q11.2 microdeletion].

OBJECTIVE: To analyze a child with facial abnormalities with combined cytogenetic and molecular techniques and delineate its clinical phenotype. METHODS: Neuropsychological profile of the child was analyzed. Color Doppler, CT and MRI were used for detecting the nodules in the body. Conventional peripheral blood karyotypes of the child and his parents were analyzed with G-banding. Array-comparative genomic hybridization (aCGH) was performed to detect minor structural chromosomal abnormalities. RESULTS: The child had mental retardation, maxillofacial dysmorphism on the right side, and irregular solid nodules on the back. The karyotypes of the child and his parents were all normal, while aCGH has identified a de novo constitutive 1.2 Mb deletion at 17q11.2 in the child. The aCGH results of his parents were normal. CONCLUSION: The de novo 17q11.2 microdeletion probably underlies the facial abnormalities and neurofibromatosis in the patient.

[Analysis of PRRT2 gene mutations in a Chinese family affected with paroxysmal kinesigenic dyskinesia].

OBJECTIVE: To screen potential mutations of PRRT2 gene in a Chinese family affected with paroxysmal kinesigenic dyskinesia (PKD). METHODS: Polymerase chain reaction, DNA sequencing and restriction endonuclaese analysis were used to analyze all members of the family. RESULTS: A heterozygous mutation c.649dupC was identified in the PRRT2 gene in all patients, while no similar mutation was found in healthy members from the family. CONCLUSION: The c.649dupC mutation of the PRRT2 gene probably underlies the PKD in this family. Prenatal diagnosis can reduce the risk for further birth of affected children for this family.

[Phenotypic and genetic analysis of a child featuring multiple malformations due to chromosome 14q deletion].

OBJECTIVE: To analyze a child with mental retardation, growth retardation and language development disorders. METHODS: Conventional G-banding analysis was performed on chromosomes cultivated from peripheral blood samples derived from the child and her parents. Array-comparative genomic hybridization (aCGH) was performed to detect minor structural chromosomal abnormalities, and the result was confirmed by short tandem repeats (STR) analysis. RESULTS: For the child and her parents, no karyotypic abnormality was detected. However, aCGH analysis has identified a 14q22.1 deletion in the child. The microdeletion, with a size of 2.9 Mb was confirmed by STR analysis. CONCLUSION: The 2.9 Mb chromosomal microdeletion probably underlies the mental retardation, growth retardation and language development disorders in the child.

[Phenotypic and genetic analysis of a girl with multiple congenital deformities due to 2p15-p16.1 microdeletion syndrome].

OBJECTIVE: To determine the origin of chromosomal aberration for a girl with mental retardation and multiple congenital deformities. METHODS: The karotypes of the girl and her parents were analyzed with routine G-banding .Their genomic DNA was also analyzed with array comparative genomic hybridization (aCGH). Short tandem repeats (STR) were used to confirm the results of aCGH. RESULTS: There were no karyotypic abnormality detected at cytogenetic level. aCGH identified a de novo 1.28 Mb deletion at 2p15-p16.1 in the girl. The results of the STR confirmed the deletion affected the maternal chromosome. CONCLUSION: The de novo interstitial 2p15-p16.1 deletion may cause the mental retardation and multiple congenital deformities. chr2:60.5-61.5 Mb may be the minimal common region of 2p15-p16.1 microdeletion syndrome.

[Mutation analysis and prenatal diagnosis of COL1A1 gene in a Chinese family with type I osteogenesis imperfecta].

OBJECTIVE: To detect mutation of COL1A1 gene in a Chinese family affected with type I osteogenesis imperfecta (OI) and to provide prenatal diagnosis for a fetus at 17th gestational week. METHODS: Polymerase chain reaction, DNA sequencing and restriction endonuclease analysis were used to verify the detected mutation among other members of the family and 100 healthy controls. RESULTS: No mutation has been detected in the COL1A2 gene in all of the subjects. A heterozygous mutation c.104-1G>C was identified in the COL1A1 gene among all patients from this family. The same mutation was not found in other members from the family and the 100 healthy controls. The mutation was not found in the fetus, and was verified to be a new mutation according to the type I collagen mutation database. CONCLUSION: The c.104-1G>C mutation of the COL1A1 gene probably underlies the type I osteogenesis imperfecta in this family. Under the premise of a clear genetic diagnosis, prenatal diagnosis may be provided to reduce the risk for the disease.