OBJECTIVE: To assess the value of genetic screening by high-throughput sequencing (HTS) for the early diagnosis of neonatal diseases. METHODS: A total of 2 060 neonates born at Ningbo Women and Children's Hospital from March to September 2021 were selected as the study subjects. All neonates had undergone conventional tandem mass spectrometry metabolite analysis and fluorescent immunoassay analysis. HTS was carried out to detect the definite pathogenic variant sites with high-frequency of 135 disease-related genes. Candidate variants were verified by Sanger sequencing or multiplex ligation-dependent probe amplification (MLPA). RESULTS: Among the 2 060 newborns, 31 were diagnosed with genetic diseases, 557 were found to be carriers, and 1 472 were negative. Among the 31 neonates, 5 had G6PD, 19 had hereditary non-syndromic deafness due to variants of GJB2, GJB3 and MT-RNR1 genes, 2 had PAH gene variants, 1 had GAA gene variants, 1 had SMN1 gene variants, 2 had MTTL1 gene variants, and 1 had GH1 gene variants. Clinically, 1 child had Spinal muscular atrophy (SMA), 1 had Glycogen storage disease II, 2 had congenital deafness, and 5 had G6PD deficiency. One mother was diagnosed with SMA. No patient was detected by conventional tandem mass spectrometry. Conventional fluorescence immunoassay had revealed 5 cases of G6PD deficiency (all positive by genetic screening) and 2 cases of hypothyroidism (identified as carriers). The most common variants identified in this region have involved DUOX2 (3.93%), ATP7B (2.48%), SLC26A4 (2.38%), GJB2 (2.33%), PAH (2.09%) and SLC22A5 genes (2.09%). CONCLUSION Neonatal genetic screening has a wide range of detection and high detection rate, which can significantly improve the efficacy of newborn screening when combined with conventional screening and facilitate secondary prevention for the affected children, diagnosis of family members and genetic counseling for the carriers.
Child ; Infant, Newborn ; Humans ; Female ; Prospective Studies ; Connexins/genetics* ; Connexin 26/genetics* ; Glucosephosphate Dehydrogenase Deficiency ; Mutation ; Sulfate Transporters/genetics* ; DNA Mutational Analysis ; Genetic Testing/methods* ; Deafness/genetics* ; Neonatal Screening/methods* ; Hearing Loss, Sensorineural/genetics* ; High-Throughput Nucleotide Sequencing ; Solute Carrier Family 22 Member 5/genetics*

Objective: To clarify the phenotypes of the newborns with SLC26A4 single-allele mutation in deafness genetic screening and second variant; to analyze the SLC26A4 genotype and hearing phenotype. Methods: 850 newborns born in Beijing from April 2015 to December 2019 were included and there were 468 males and 382 females. They received genetic deafness screening for 9 or 15 variants, with the result of SLC26A4 single-allele mutation. Firstly, three step deafness gene sequencing was adopted in this work, i.e., the first step was "SLC26A4 gene whole exons and splice sites" sequencing; the second step was "SLC26A4 gene promoter, FOXI1 gene and KCNJ10 gene whole exons" sequencing; and the third step was detection for "SLC26A4 gene copy number variation". Secondly, we collected the results of newborn hearing screening for all patients with the second mutation found in the three step test, and conducted audiological examinations, such as acoustic immittance, auditory brainstem response and auditory steady state response. Thirdly, for novel/VUS mutations, we searched the international deafness gene database or software, such as DVD, ClinVar and Mutation Taster, to predict the pathogenicity of mutations according to the ACMG guideline. Lastly, we analyzed the relationship between genotype and phenotype of newborns with SLC26A4 single allele mutation. Results: Among 850 cases, the median age of diagnosis was 4 months. In the first step, 850 cases were sequenced. A total of 32 cases (3.76%, 32/850) of a second variants were detected, including 18 cases (2.12%, 18/850) with identified pathogenic variants; 832 cases were sequenced and 8 cases of KCNJ10 gene missense variants were detected among the second step. No missense mutations in the FOXI1 gene and abnormal SLC26A4 gene promoter were detected; the third step sequencing results were all negative. Genotypes and hearing phenotypes included 18 cases combined with the second clear pathogenic variant, 16 cases (16/18) referred newborn hearing screening and 2 cases (2/18) passed in both ears; degree of hearing loss consisted of 18 profound ears (18/36), 13 severe ears (13/36) and 5 moderate ears (5/36); audiogram patterns comprised 17 high frequency drop ears (17/36), 14 flat ears (14/36), 3 undistinguished ears (3/36), and 2 U shaped ears (2/36); 11 cases underwent imaging examination, all of which were bilateral enlarged vestibular aqueduct. As for 22 cases of other genotypes, all passed neonatal hearing screening and the hearing diagnosis was normal, including 9 cases with VUS or possibly novel benign variants, 8 cases with KCNJ10 double gene heterozygous variants, and 5 cases with double heterozygous variants. Conclusions: The probability of individuals with SLC26A4 single-allele variant who merge with a second pathogenic variant is 2.12%, all of which are SNV, which can provide scientific basis for the genetic diagnosis and genetic counseling of SLC26A4 variants. Those who have merged with second pathogenic variant are all diagnosed with sensorineural hearing loss. Patients with KCNJ10 gene mutations do not manifest hearing loss during the infancy, suggesting the need for further follow-up.
Female ; Humans ; Male ; Alleles ; Deafness/genetics* ; DNA Copy Number Variations ; Forkhead Transcription Factors/genetics* ; Genotype ; Hearing Loss/genetics* ; Hearing Loss, Sensorineural/genetics* ; Mutation ; Phenotype ; Sulfate Transporters/genetics* ; Vestibular Aqueduct ; Infant, Newborn ; Potassium Channels, Inwardly Rectifying/genetics*

Antiporters/genetics* ; Carcinoma, Papillary/pathology* ; Humans ; Neoplasm Metastasis/genetics* ; Sulfate Transporters/genetics* ; Thyroid Cancer, Papillary/pathology* ; Thyroid Neoplasms/pathology*

OBJECTIVE: To detect additional variants for newborn carriers of single heterozygous variants of the GJB2 or SLC26A4 gene by genechip analysis in Changsha area, and explore the variation spectrum of deafness-related genes in this region. METHODS: For 462 newborns carrying single heterozygous variants of the GJB2 or SLC26A4 gene, all exons of the genes were subjected to Sanger sequencing. The pathogenicity of the variants was analyzed by database and literature search. RESULTS: For 305 newborns carrying a heterozygous GJB2 variant, 143 (46.49%) were found to carry additional variants, including 29 (9.51%) with c.109G>A likely pathogenic variant, and 1 (6.48%) with c.551G>A pathogenic variant. Among 153 newborns carrying single heterozygous variant of the SLC26A4 gene, 2 (1.31%) were found with a c.281C>T variant, and 1 (0.65%) with a c.1547_1548ins pathogenic variant. Among 4 newborns simultaneously carrying GJB2 and SLC26A4 variants, two were found to carry c.109G>A and c.844T>C variants (clinical significance unknown), respectively. CONCLUSION For newborns carrying single heterozygous variants of the GJB2 or SLC26A4 gene by genechip analysis, the detection rate for other variants is quite high. Sanger sequencing can significantly improve the detection rate of high-risk newborns and enrich the variant spectrum of deafness genes.
Connexins/genetics* ; DNA Mutational Analysis ; Deafness/genetics* ; Genetic Carrier Screening ; Heterozygote ; Humans ; Infant, Newborn ; Mutation ; Oligonucleotide Array Sequence Analysis ; Sulfate Transporters/genetics*

OBJECTIVE: To determine the frequencies of deafness gene mutations among patients with non-syndromic hearing loss (NSHL) from northern Jiangsu province. METHODS: A total of 117 patients with NSHL were enrolled. The coding region of GJB2 gene, IVS7-2A>G and 2168A>G mutations of SLC26A4 gene, and 1555A>G and 1494C>T mutations of mitochondrial DNA 12S rRNA were subjected to Sanger sequencing. Patients in whom no mutation was detected were further tested by targeted gene capture and high-throughput sequencing. RESULTS: Among the 117 patients, 86 (73.50%) were found to carry mutations. GJB2 gene mutations were found in 61 patients (52.14%), including 22 (18.80%) with homozygous mutations and 39 (33.33%) with heterozygous mutations. SLC26A4 gene mutations were found in 19 patients (16.24%), including 4 (3.42%) with homozygous mutations and 15 with heterozygous mutations (14.53%). Mitochondrial 12S rRNA gene mutation was found in 6 patients (5.13%). Targeted gene capture and high-throughput sequencing of 8 patients identified 4 further cases, including 1 with RDX gene 129_130del and 76_79del compound heterozygous mutations, 1 with OTOF gene 1274G>C homozygous mutation, 1 with SLC26A4 gene 919-2A>G and IVS16-6G>A compound heterozygous mutation, and 1 with SLC26A4 gene 919-2A>G and A1673T compound heterozygous mutation. CONCLUSION The frequency of mutation among patients with NSHL from north Jiangsu was 73.50%, and GJB2 gene was most commonly mutated.
China ; Connexins ; DNA Mutational Analysis ; DNA, Mitochondrial ; Hearing Loss ; genetics ; Humans ; Membrane Proteins ; Mutation ; Sulfate Transporters

OBJECTIVE: To establish a congenital chloride diarrhea (CCD)-associated SLC26A3 c.392C>G (p.P131R) polymorphism-expressing cell model, and to investigate its biological function. METHODS: The sequence of the SLC26A3 gene in GenBank was used to design the upstream and downstream single-guide RNA (sgRNA) that could specifically recognize the 392 locus of the SLC26A3 gene, and the sgRNA was mixed with the pSpCas9-puro vector after enzyme digestion to construct an eukaryotic recombinant expression plasmid (pSpCas9-SLC26A3). Caco-2 cells were transfected with the recombinant plasmid and synthesized single-stranded DNA oligonucleotides (ssODNs), and Taqman genotyping assay and Sanger sequencing were used to identify the expression of SLC26A3 c.392C>G (p.P131R) in Caco-2 cells. Wild-type Caco-2 cells were selected as normal control group and the Caco-2 cells with successful expression of SLC26A3 c.392C>G (p.P131R) was selected as P131R group. Both groups were treated with 100 ng/mL tumor necrosis factor-α (TNF-α), and then the normal control group was named as TNF-α group, and the P131R group was named as TNF-α+P131R group. Electric cell-substrate impedance sensing (ECIS) assay was used to evaluate the change in the monolayer barrier function of intestinal epithelial cells in the above four groups, and Western blot was used to measure the change in the expression of SLC26A3 protein in the normal control group and the P131R group. RESULTS: The eukaryotic recombinant expression plasmid (pSpCas9-SLC26A3) was successfully constructed. Both Taqman genotyping assay and Sanger sequencing confirmed the successful establishment of the Caco-2 cell model of SLC26A3 c.392C>G (p.P131R) expression. ECIS assay showed that compared with the normal control group, the P131R group had a significant increase in the monolayer permeability of intestinal epithelial cells (P<0.05), and at the same time, the P131R group had a significantly greater increase in cell membrane permeability after the induction with 100 ng/mL TNF-α (P<0.05). Western blot showed that compared with the normal control group, the P131R group had a significant reduction in the expression of SLC26A3 protein (P=0.001). CONCLUSIONS SLC26A3 c.392C>G (p.P131R) can reduce the expression of SLC26A3 protein, increase the monolayer permeability of intestinal epithelial cells, and thus lead to diarrhea.
Caco-2 Cells ; Chloride-Bicarbonate Antiporters ; genetics ; Diarrhea ; congenital ; genetics ; Humans ; Intestinal Mucosa ; Metabolism, Inborn Errors ; genetics ; Polymorphism, Single Nucleotide ; Sulfate Transporters ; genetics ; Tight Junctions ; Tumor Necrosis Factor-alpha

Hepatitis B virus (HBV) is the prototype of hepatotropic DNA viruses (hepadnaviruses) infecting a wide range of human and non-human hosts. Previous studies with duck hepatitis B virus (DHBV) identified duck carboxypeptidase D (dCPD) as a host specific binding partner for full-length large envelope protein, and p120 as a binding partner for several truncated versions of the large envelope protein. p120 is the P protein of duck glycine decarboxylase (dGLDC) with restricted expression in DHBV infectible tissues. Several lines of evidence suggest the importance of dCPD, and especially p120, in productive DHBV infection, although neither dCPD nor p120 cDNA could confer susceptibility to DHBV infection in any cell line. Recently, sodium taurocholate cotransporting polypeptide (NTCP) has been identified as a binding partner for the N-terminus of HBV large envelope protein. Importantly, knock down and reconstitution experiments unequivocally demonstrated that NTCP is both necessary and sufficient for in vitro infection by HBV and hepatitis delta virus (HDV), an RNA virus using HBV envelope proteins for its transmission. What remains unclear is whether NTCP is the major HBV receptor in vivo. The fact that some HBV patients are homozygous with an NTCP mutation known to abolish its receptor function suggests the existence of NTCP-independent pathways of HBV entry. Also, NTCP very likely mediates just one step of the HBV entry process, with additional co-factors for productive HBV infection still to be discovered. NTCP offers a novel therapeutic target for the control of chronic HBV infection.
Animals ; Carboxypeptidases/genetics/*metabolism ; Gene Products, pol/genetics/metabolism ; Heparan Sulfate Proteoglycans/metabolism ; Hepatitis B virus/*physiology ; Hepatocytes/metabolism/virology ; Organic Anion Transporters, Sodium-Dependent/antagonists & inhibitors/genetics/metabolism ; RNA Interference ; Symporters/antagonists & inhibitors/genetics/metabolism ; Viral Envelope Proteins/metabolism ; Virus Internalization

OBJECTIVE: To preliminarily determine the gene mutation frequency and the hotspots in Henan province, we analysed the deafness-related gene mutation in patients with non-syndromic hearing loss (NSHL). METHOD: Genomic DNA samples of 100 patients with NSHL in Henan province were extracted from peripheral blood after clinical history inquiry and clinical examination, Four common deafness genes GJB2, SLC26A4, mitochondrial 12SrRNA, and GJB3 were detected by Sanger sequencing method,and then data analysis were conducted. RESULT: Among 100 patients with NSHL. the gene mutation frequency was 44%. In these patients, 29 cases had GJB2 mutations, 13 cases had SLC26A4 gene mutations, and 3 cases had mitochondrial 12SrRNA mutations. CONCLUSION Among the patients with NSHL in Henan province, the most frequent mutation causing hereditary deafness was mutation in GJB2, followed by SLC26A4,and it will provide a theoretical basis to determine the etiology of deafness in Henan Province.
China ; Connexin 26 ; Connexins ; genetics ; DNA Mutational Analysis ; DNA, Mitochondrial ; genetics ; Deafness ; genetics ; Gene Frequency ; Humans ; Membrane Transport Proteins ; genetics ; Mutation ; RNA, Ribosomal ; genetics ; Sulfate Transporters

OBJECTIVE: To investigate the mutation characteristics of common deafness gene from 127 non-syndromic hearing loss patients in Guangxi province. METHOD: Deafness-related gene mutations detection kit was used to detect 15 mutation sites in four deafness-associated genes, and a total of 127 hearing impaired patients were tested. The samples that could not be diagnosed with DNA microarray were subjected to PCR and sequenced to detect other mutations. RESULT Among the 127 patients with non-syndromic deafness, the total mutation rate is 8.66% (11/127), including GJB2 235delC homozygous in 3 cases (2.36%), 235delC single heterozygous mutation in 2 cases (1.57%), GJB2 235delC and 109 A > G mutations in 2 cases (1.57%); SLC26A4 1229C > T homozygous in 1 case (0.79%), IVS7-2A > G, IVS11 + 47T > C and 15448insC mutaion in 2 cases (1.57%); mitochondrial 12S rRNA gene mutations were not detected. The result indicates that GJB2 and SLC26A4 were the main genes in this study, and the mutation rate is significantly lower than the national average level. Three new mutations (SLC26A4 IVS11 + 47T > C,1548insC and GJB2 109A > G) were found. There may be rare mutations among sites or genes associated with deafness in Guangxi.
China ; Connexin 26 ; Connexins ; genetics ; DNA Mutational Analysis ; Deafness ; genetics ; Heterozygote ; Homozygote ; Humans ; Membrane Transport Proteins ; genetics ; Mutation ; Oligonucleotide Array Sequence Analysis ; Polymerase Chain Reaction ; RNA, Ribosomal ; genetics ; Sulfate Transporters

OBJECTIVE: To summarize and analyze the clinical characteristics of Mondini dysplasia with cerebrospinal fluid leakage, as well as preliminarily investigate the genetic mechanism of the disease. METHOD: The clinical data of 2 patients diagnosed as Mondini dysplasia with cerebrospinal fluid leakage treated in our hospital were analyzed. Blood samples of these two patients were obtained to extract DNA. We screened DNA samples for gene SLC26A4 mutations by using polymerase chain reaction and direct sequencing. The sequencing results were analyzed in DNASTAR software. RESULT: Both patients came to our hospital because of recurrent meningitis, and the fistula were both located in vestibular window. Patients were cured one-time after surgical closure of the leakages with temporalis + temporalis fascia + temporalis through the mastoid approach. No pathogenic mutations of gene SLC26A4 with exome sequencing were found. CONCLUSION Mondini dysplasia with cerebrospinal fluid leakage should be considered in patients with recurrent meningitis and hearing disorder. Temporal bone HRCT is helpful to the diagnosis. Surgical closure is an effective therapeutic method and may prevent recurrent meningitis. The molecular mechanism of simple Mondini dysplasia needs further study.
Cerebrospinal Fluid Leak ; physiopathology ; Cochlea ; pathology ; Fistula ; pathology ; Humans ; Hyperplasia ; genetics ; physiopathology ; Membrane Transport Proteins ; genetics ; Meningitis ; physiopathology ; Mutation ; Sulfate Transporters