Systemic immune profiling of Omicron-infected subjects inoculated with different doses of inactivated virus vaccine.

SARS-CoV-2 primary strain-based vaccination exerts a protective effect against Omicron variants-initiated infection, symptom occurrence, and disease severity in a booster-dependent manner. Yet, the underlying mechanisms remain unclear. During the 2022 Omicron outbreak in Shanghai, we enrolled 122 infected adults and 50 uninfected controls who had been unvaccinated or vaccinated with two or three doses of COVID-19 inactive vaccines and performed integrative analysis of 41-plex CyTOF, RNA-seq, and Olink on their peripheral blood samples. The frequencies of HLA-DRhi classical monocytes, non-classical monocytes, and Th1-like Tem tended to increase, whereas the frequency of Treg was reduced by booster vaccine, and they influenced symptom occurrence in a vaccine dose-dependent manner. Intercorrelation and mechanistic analysis suggested that the booster vaccination induced monocytic training, which would prime monocytic activation and maturation rather than differentiating into myeloid-derived suppressive cells upon Omicron infections. Overall, our study provides insights into how booster vaccination elaborates protective immunity across SARS-CoV-2 variants.

Identification of the ataxin-1 interaction network and its impact on spinocerebellar ataxia type 1.

BACKGROUND: Spinocerebellar ataxia type 1 (SCA1) is a neurodegenerative disease caused by a polyglutamine expansion in the ataxin-1 protein. The pathogenic mechanism resulting in SCA1 is still unclear. Protein-protein interactions affect the function and stability of ataxin-1. METHODS: Wild-type and mutant ataxin-1 were expressed in HEK-293T cells. The levels of expression were assessed using real-time polymerase chain reaction (PCR) and Western blots. Co-immunoprecipitation was done in HEK-293T cells expressing exogenous wild-type and mutant ataxin-1 using anti-Flag antibody following by tandem affinity purification in order to study protein-protein interactions. The candidate interacting proteins were validated by immunoprecipitation. Chromatin immunoprecipitation and high-throughput sequencing and RNA immunoprecipitation and high-throughput sequencing were performed using HEK-293T cells expressing wild-type or mutant ataxin-1. RESULTS: In this study using HEK-293T cells, we found that wild-type ataxin-1 interacted with MCM2, GNAS, and TMEM206, while mutant ataxin-1 lost its interaction with MCM2, GNAS, and TMEM206. Two ataxin-1 binding targets containing the core GGAG or AAAT were identified in HEK-293T cells using ChIP-seq. Gene Ontology analysis of the top ataxin-1 binding genes identified SLC6A15, NTF3, KCNC3, and DNAJC6 as functional genes in neurons in vitro. Ataxin-1 also was identified as an RNA-binding protein in HEK-293T cells using RIP-seq, but the polyglutamine expansion in the ataxin-1 had no direct effects on the RNA-binding activity of ataxin-1. CONCLUSIONS: An expanded polyglutamine tract in ataxin-1 might interfere with protein-protein or protein-DNA interactions but had little effect on protein-RNA interactions. This study suggested that the dysfunction of protein-protein or protein-DNA interactions is involved in the pathogenesis of SCA1.

Mutations in C1orf194, encoding a calcium regulator, cause dominant Charcot-Marie-Tooth disease.

Charcot-Marie-Tooth disease is a hereditary motor and sensory neuropathy exhibiting great clinical and genetic heterogeneity. Here, the identification of two heterozygous missense mutations in the C1orf194 gene at 1p21.2-p13.2 with Charcot-Marie-Tooth disease are reported. Specifically, the p.I122N mutation was the cause of an intermediate form of Charcot-Marie-Tooth disease, and the p.K28I missense mutation predominately led to the demyelinating form. Functional studies demonstrated that the p.K28I variant significantly reduced expression of the protein, but the p.I122N variant increased. In addition, the p.I122N mutant protein exhibited the aggregation in neuroblastoma cell lines and the patient's peroneal nerve. Either gain-of-function or partial loss-of-function mutations to C1ORF194 can specify different causal mechanisms responsible for Charcot-Marie-Tooth disease with a wide range of clinical severity. Moreover, a knock-in mouse model confirmed that the C1orf194 missense mutation p.I121N led to impairments in motor and neuromuscular functions, and aberrant myelination and axonal phenotypes. The loss of normal C1ORF194 protein altered intracellular Ca2+ homeostasis and upregulated Ca2+ handling regulatory proteins. These findings describe a novel protein with vital functions in peripheral nervous systems and broaden the causes of Charcot-Marie-Tooth disease, which open new avenues for the diagnosis and treatment of related neuropathies.

A multi-stage association study of plasma cytokines identifies osteopontin as a biomarker for acute coronary syndrome risk and severity.

Cytokines play a critical role in the pathogenesis and development of cardiovascular diseases. However, data linking cytokines to risk and severity of acute coronary syndrome (ACS) are still limited. We measured plasma profile of 280 cytokines using a quantitative protein microarray in 12 ACS patients and 16 healthy controls, and identified 15 differentially expressed cytokines for ACS. Osteopontin, chemokine ligand 23, brain derived neurotrophic factor and C-reactive protein (CRP) were further validated using immunoassay in two independent case-control studies with a total of 210 ACS patients and 210 controls. We further examined their relations with incident ACS among 318 case-control pairs nested within the Dongfeng-Tongji cohort, and found plasma osteopontin and CRP concentrations were associated with incident ACS, and the multivariable-adjusted odds ratio (95% confidence interval) was 1.29 (1.06-1.57) per 1-SD increase for osteopontin and 1.30 (1.02-1.66) for CRP, respectively. Higher levels of circulating osteopontin were also correlated with higher severity of ACS, and earlier ACS onset time. Adding osteopontin alone or in combination with CRP modestly improved the predictive ability of ACS beyond the Framingham risk scores. Our findings suggested that osteopontin might be a biomarker for incident ACS, using osteopontin adds moderately to traditional cardiovascular risk factors.

Diagnosis of polyglutamine spinocerebellar ataxias by polymerase chain reaction amplification and Sanger sequencing.

Spinocerebellar ataxia (SCA) is a group of genetic diseases of the nervous system with genetic and clinical heterogeneity. SCA is often caused by an expanded CAG repeat sequence in the encoding protein. Genetic testing is necessary to diagnose and classify the types of SCA. Next­generation DNA sequencing usually generates a high error rate for insertion or deletion mutations, so it is unhelpful for classifying the types of SCA. In the present study, a Chinese SCA pedigree was preliminarily diagnosed with SCA1 using polymerase chain reaction (PCR) amplification. The propositus and his three younger siblings were diagnosed with SCA1 as a result of the identification of the length of the expanded CAG repeat sequence in the ATXN1 gene performed using Sanger sequencing. The current study presents a convenient and efficient method to identify causative mutations for polyglutamine SCA using PCR amplification followed by Sanger sequencing.

Genome-Wide Analysis of DNA Methylation and Acute Coronary Syndrome.

RATIONALE: Acute coronary syndrome (ACS) is a leading cause of death worldwide. Immune functions play a vital role in ACS development; however, whether epigenetic modulation contributes to the regulation of blood immune cells in this disease has not been investigated. OBJECTIVE: We conducted an epigenome-wide analysis with circulating immune cells to identify differentially methylated genes in ACS. METHODS AND RESULTS: We examined genome-wide methylation of whole blood in 102 ACS patients and 101 controls using HumanMethylation450 array, and externally replicated significant discoveries in 100 patients and 102 controls. For the replicated loci, we further analyzed their association with ACS in 6 purified leukocyte subsets, their correlation with the expressions of annotated genes, and their association with cardiovascular traits/risk factors. We found novel and reproducible association of ACS with blood methylation at 47 cytosine-phosphoguanine sites (discovery: false discovery rate <0.005; replication: Bonferroni corrected P<0.05). The association of methylation levels at these cytosine-phosphoguanine sites with ACS was further validated in at least 1 of the 6 leukocyte subsets, with predominant contributions from CD8+ T cells, CD4+ T cells, and B cells. Blood methylation of 26 replicated cytosine-phosphoguanine sites showed significant correlation with expressions of annotated genes (including IL6R, FASLG, and CCL18; P<5.9×10-4), and differential gene expression in case versus controls corroborated the observed differential methylation. The replicated loci suggested a role in ACS-relevant functions including chemotaxis, coronary thrombosis, and T-cell-mediated cytotoxicity. Functional analysis using the top ACS-associated methylation loci in purified T and B cells revealed vital pathways related to atherogenic signaling and adaptive immune response. Furthermore, we observed a significant enrichment of the replicated cytosine-phosphoguanine sites associated with smoking and low-density lipoprotein cholesterol (Penrichment≤1×10-5). CONCLUSIONS: Our study identified novel blood methylation alterations associated with ACS and provided potential clinical biomarkers and therapeutic targets. Our results may suggest that immune signaling and cellular functions might be regulated at an epigenetic level in ACS.

Genome-Wide Analysis of DNA Methylation and Cigarette Smoking in a Chinese Population.

BACKGROUND: Smoking is a risk factor for many human diseases. DNA methylation has been related to smoking, but genome-wide methylation data for smoking in Chinese populations is limited. OBJECTIVES: We aimed to investigate epigenome-wide methylation in relation to smoking in a Chinese population. METHODS: We measured the methylation levels at > 485,000 CpG sites (CpGs) in DNA from leukocytes using a methylation array and conducted a genome-wide meta-analysis of DNA methylation and smoking in a total of 596 Chinese participants. We further evaluated the associations of smoking-related CpGs with internal polycyclic aromatic hydrocarbon (PAH) biomarkers and their correlations with the expression of corresponding genes. RESULTS: We identified 318 CpGs whose methylation levels were associated with smoking at a genome-wide significance level (false discovery rate < 0.05), among which 161 CpGs annotated to 123 genes were not associated with smoking in recent studies of Europeans and African Americans. Of these smoking-related CpGs, methylation levels at 80 CpGs showed significant correlations with the expression of corresponding genes (including RUNX3, IL6R, PTAFR, ANKRD11, CEP135 and CDH23), and methylation at 15 CpGs was significantly associated with urinary 2-hydroxynaphthalene, the most representative internal monohydroxy-PAH biomarker for smoking. CONCLUSION: We identified DNA methylation markers associated with smoking in a Chinese population, including some markers that were also correlated with gene expression. Exposure to naphthalene, a byproduct of tobacco smoke, may contribute to smoking-related methylation. CITATION: Zhu X, Li J, Deng S, Yu K, Liu X, Deng Q, Sun H, Zhang X, He M, Guo H, Chen W, Yuan J, Zhang B, Kuang D, He X, Bai Y, Han X, Liu B, Li X, Yang L, Jiang H, Zhang Y, Hu J, Cheng L, Luo X, Mei W, Zhou Z, Sun S, Zhang L, Liu C, Guo Y, Zhang Z, Hu FB, Liang L, Wu T. 2016. Genome-wide analysis of DNA methylation and cigarette smoking in Chinese. Environ Health Perspect 124:966-973; http://dx.doi.org/10.1289/ehp.1509834.

[Pathogenic Genes of A Hereditary Hemorrhagic Telangiectasia Pedigree].

OBJECTIVE: To identify the mutation of ENG and ALK1 genes in a hereditary hemorrhagic telangiectasia pedigree. METHODS: 14 exons of ENG gene and 9 exons of ALK1 gene in 11 menbers of this pedigree 4 generation were amplified by reverse transcription-polymerase chain reaction (RT-PCR), the PCR products were screened by direct sequencing. RESULTS: A nonsense mutation c.447G > A was found in exon 4 of ENG gen of the pedigreee, resulting in change of Trp 149 into Stop, while no gene mutation was found in ALK1 gene. CONCLUSION: The hereditary hemorrhagic telangiectasia in this pedigree is caused by the nonsense mutation c.447G > A in ENG gene.

A complex insertion/deletion polymorphism in the compositionally biased region of the ZFHX3 gene in patients with coronary heart disease in a Chinese population.

Coronary heart disease (CHD) is a leading cause of morbidity and mortality around the world and has both genetic and environmental precipitants. Genetic factors are significant in determining the level of risk factors in individuals. Variants in ZFHX3 gene are associated with atrial fibrillation in individuals of European ancestry. The aim of this study was to analyze the polymorphisms in the compositionally biased region of the ZFHX3 gene in patients with coronary heart disease in a Chinese population, and to explore their associations with coronary heart disease. We recruited 278 CHD patients and 358 age and sex matched healthy controls in a Chinese Han population, polymorphisms in the compositionally biased region of the ZFHX3 gene were determined by polymerase chain reaction followed by DNA sequencing. The genotype frequencies were calculated, and statistical analysis was performed using the non-parametric mood median test. A complex insertion/deletion polymorphism was identified in the compositionally biased region of the ZFHX3 gene in a Chinese population. Six common genotypes (GGC)4GGTGGCAGT(GGC)4GGT(GGC)8, (GGC)4GGTGGCAGT(GGC)5GGT(GGC)8, (GGC)4GGTGGCAGT(GGC)5GGT(GGC)7, (GGC)2GGTGGCAGT(GGC)5GGT(GGC)10, (GGC)4GGTGGCAGT(GGC)5GGT(GGC)5, and (GGC)4GGT(GGC)8 were found in both CHD patients and healthy controls, there was no significant difference in the six genotype frequencies between CHD patients and healthy controls. Rare genotypes (GGC)4GGTGGCAGT(GGC)2GGT(GGC)2GGT(GGC)6, (GGC)4GGTGGCAGT (GGC)8, (GGC)4GGTGGCAGT(GGC)(3)GGT(GGC)8, and (GGC)6GGT(GGC)8 were only identified in healthy controls. Rare genotypes (GGC)4GGTGGCAGT(GGC)4GGT(GGC)5, (GGC)4GGTGGCAGT(GGC)4GGT(GGC)4, and (GGC)4GGTGGCGGT(GGC)6 were only found in CHD patients. The compositionally biased region of the ZFHX3 gene contains a poly-Gly sequence. A complex insertion/deletion polymorphism exists in this region in a Chinese population, clinical significance of some rare genotypes should be explored for CHD in the future.

[Sequence Analysis of the UGT1A1 Gene Untranslated Region in Chinese Han Population].

OBJECTIVE: To analyze the 5' and 3'-untranslated region sequences of the UGT1A1 gene in Chinese Han population and to find polymorphic variants within the untranslated region. METHODS: Genomic DNA was extracted from peripheral leukocytes in 220 healthy Han individuals. The 5' and 3'-untranslated region sequences of the UGT1A1 gene were amplified by polymerase chain reaction, and followed by DNA sequencing. RESULTS: Two polymorphic loci were identified in the 5'-untranslated region of the UGT1A1 gene with -64(G/C) and A(TA)6TAA/A(TA)7TAA in TATAA box region among Chinese Han population. Genotype frequencies were 98.4% (G) and 1.6% (C) in -64 locus of the UGT1A1 gene among the 220 individuals. The allele frequency of A(TA)6TAA and A(TA)7TAA within the promoter region was found to be 93.4% and 6.6%, respectively. Two polymorphic loci of 1813(C/T) and 1941(C/G) were detected in the 3'-untranslated region of the UGT1A1 gene, they showed a homozygous state at two loci with cosegregation pattern at 1813 and 1941 locus. The haplotype frequencies were 73.6% (CC/1813+CC/1941) and 26.4% (TT/1813+GG/1941) for 1813 and 1941 loci in the UGT1A1 gene. CONCLUTION: Cosegregation pattern, at 1813 and 1941 locus with homozygous state in the 3'-untranslated region of the UGT1A1 gene may be selected from the human genome among Chinese Han population. More studies should be focused on the mechanism of homozygous cosegregation.

The CAA repeat polymorphism in the ZFHX3 gene is associated with risk of coronary heart disease in a Chinese population.

Coronary heart disease (CHD) is a disease resulting from the interaction between genetic variations and environmental factors. Zinc finger homeobox 3 (ZFHX3) is a transcription factor and contains a poly-glutamine tract in a compositionally biased region that is encoded by exon 9, containing a cluster of CAG and CAA triplets followed by the polymorphic CAA repeats: (CAG)2(CAA)2(CAG)3CAACAG(CAA)nGCA. Thus, nine successive glutamine residues precede the poly-glutamine tract, encoded by the polymorphic CAA repeats. The aim of this study was to investigate the association of the CAA repeat polymorphism in exon 9 of the ZFHX3 gene with the risk of CHD in a Chinese population. The CAA repeat polymorphism was determined by polymerase chain reaction followed by DNA sequencing in 321 CHD patients. Genotype frequencies were compared using the non-parametric mood median test. Four alleles of CAG(CAA)10GCA, CAG(CAA)8GCA, CAG(CAA)9GCA, and CAG(CAA)11GCA were found in Chinese CHD patients in exon 9 of the ZFHX3 gene. The CAG(CAA)10GCA was a major allele (95.95%), and the CAG(CAA)8GCA was a minor allele (3.58%). The CAG(CAA)9GCA and CAG(CAA)11GCA were rare alleles (0.31% and 0.16%). The CAG(CAA)10GCA allele encodes a poly-glutamine tract of 19 residues. Importantly, the CHD patients homozygous for the CAG(CAA)10GCA allele had a higher risk of CHD, compared to the heterozygous patients carrying a CAG(CAA)8GCA allele. Moreover, the CAG(CAA)10GCA allele was significantly associated with hypertension, diabetes mellitus, or dyslipidemia (P < 0.05). Thus, the CAA repeat polymorphism in exon 9 of the ZFHX3 gene contributes to the CHD susceptibility in the Chinese population.

A de novo chromosomal abnormality in Cri du Chat syndrome.

OBJECTIVE: To find the length and location of the deletions in the short arm of chromosome 5 in one case of Cri du Chat syndrome using oligo array comparative genomic hybridization. METHODS: Metaphase chromosomes were prepared from peripheral blood lymphocyte cultures using standard cytogenetic protocols. Chromosomal analysis was done in G-banded metaphases. Oligo array comparative genomic hybridization and fluorescence in situ hybridization were performed by the commercially available kits. RESULTS: Oligonucleotide array comparative genomic hybridization (CGH) analysis revealed a 23.263 Mb deletion at region 5p14.2-->qter, combined with a duplication of 14.602 Mb in size in the area 12p13.1-->pter. Chromosomal aberrations were confirmed by fluorescence in situ hybridization. The male neonate with Cri du Chat syndrome had an unbalanced translocation which was inherited from his father who was a balanced carrier with a karyotype 46, XY, t (5; 12) (p14.2; p13.1). CONCLUSIONS: This report shows the clinical utility of the oligonucleotide array in the detection of submicroscopic chromosomal aberrations, thus improving the molecular diagnosis of Cri du Chat syndrome.

[Mutation analysis of UGT1A1 gene in patients with unconjugated hyperbilirubinemia].

OBJECTIVE: To analyze potential mutations of uridine diphosphate glucuronosyltransferase 1A1 (UGT1A1) gene in patients with unconjugated hyperbilirubinemia, and to explore the correlation between the mutations and total serum bilirubin levels. METHODS: Genomic DNA was extracted from peripheral blood samples of patients. Coding sequence and promoter region of the UGT1A1 gene were amplified. Mutations were identified through DNA sequencing. RESULTS: Mutations of the UGT1A1 gene were found in 46 out of 61 patients with unconjugated hyperbilirubinemia. Five types of mutations were detected, with a decreasing order of 211G>A, TA insertion in the TATAA promoter element, 686C>A, 1091C>T and 1352C>T. Compared with those carrying a single homozygous mutation or compound heterozygous mutations, total serum bilirubin was higher in those carrying a homozygous mutation in combination with other heterozygous mutations (P< 0.05). Based on the UGT1A1 gene mutations and level of total serum bilirubin, 44 patients were diagnosed with Gilbert syndrome, and 2 were diagnosed with Crigler-Najjar syndrome type 2. CONCLUSION: The level of total serum bilirubin is correlated with the number of UGT1A1 gene mutations as well as their heterozygous or homozygous status.

[Effect of BCL11A gene on transcription of γ-globin gene].

This study was aimed to explore the effect of BCL11A gene on transcription of γ-globin gene in K562 cells. B-cell lymphoma/leukemia 11A (BCL11A) gene was silenced by small interfering RNA (siRNA) expression vectors in K562 cells (human erythroblastic leukemia cell line). Gamma-globin mRNA level in K562 cells was determined by RT-PCR. Association between the BCL11A gene and γ-globin gene transcription was explored by comparison of mRNA levels. The results indicated that the silence rate of the BCL11A gene in K562 cells by 4 siRNA expression vectors was 49.7%, 55.4%, 78.2%, and 84.1%, respectively. The siRNA expression vector with 84.1% silence rate was transfected into K562 cells, transcription level of γ-globin mRNA in K562 cells transfected with siRNA expression vector increased 2.4 times as compared with control K562 cells. It is concluded that level of γ-globin mRNA increases when the BCL11A gene is silenced. It indicates that the BCL11A gene may be a negative regulator for γ-globin gene expression.

[Correlation between hemoglobin F levels and single nucleotide polymorphism at BCL11A gene rs11886868 locus in ß-thalassemia patients].

This study was aimed to analyze hemoglobin F (HbF) level and single nucleotide polymorphisms at rs11886868 locus of BCL11A gene in ß-thalassemia patients, and to explore correlation between them. 89 mild ß-thalassemia patients with known mutations were registered, and HbF levels were determined by capillary electrophoresis. Genomic DNA was extracted from peripheral leukocytes, fragment including rs11886868 locus in BCL11A gene was amplified by PCR, and polymorphism was determined by DNA sequencing. The results showed that 2 polymorphisms including C and T were found at rs11886868 locus in BCL11A gene among 89 mild ß-thalassemia patients. HbF levels in red blood cells were (4.47 ± 3.42)% and (2.79 ± 2.21)% for ß-thalassemia patients carrying C/C and C/T haplotypes, respectively. There was difference between 2 haplotype groups. It is concluded that the C and T polymorphisms are found at rs11886868 locus in the BCL11A gene for ß-thalassemia patients. C polymorphism may be related to high HbF expression in red blood cells.

[A novel mutation in ß-globin gene of a patient with ß-thalassemia].

This study was aimed to analyze the ß-globin gene mutations in a patient with ß-thalassemia minor. Genomic DNA was extracted from peripheral blood cells of the patient. The full-length DNA sequence coding for ß-globin was amplified by polymerase chain reaction, and the gene mutation was determined by DNA sequencing. The results indicated that a heterogeneous A→G mutation was found at position 129 in intron 1 of the ß-thalassemia minor patient. It is concluded that the IVS-I-129(A→G) mutation is a splicing site mutation leading to a splicing error in immature messenger RNA and a protein translation error for the ß-globin gene. Thus, the IVS-I-129(A→G) is a novel mutation.

[Polymorphism in BP1 binding site upstream of ß-globin gene in Chinese Han population].

This study was aimed to analyze the BP1 binding site sequence upstream of ß-globin gene in Chinese Han population, and to investigate polymorphism in the BP1 binding site upstream of ß-globin gene, so as to provide the basis for exploration of relation between polymorphisms in the BP1 binding site and ß-globin expression. Genomic DNA was extracted from peripheral leukocytes of 110 healthy individuals in Chinese Han population. Sequence of the BP1 binding site upstream of ß-globin gene was amplified by polymerase chain reaction, the polymorphic variation in the BP1 binding site was determined by DNA sequencing. The results indicated that 2 polymorphism loci were found in the BP1 binding site upstream of ß-globin gene, they were C/T at the -551 bp region and (AC)(n)(AT)(x)T(y) at the -530 bp region in Chinese Han population. Frequencies of C and T were 60.4% and 39.6% at position -551. Analysis of the (AC)(n)(AT)(x)T(y) polymorphism revealed 9 different genotypes: (AC)(2)(AT)(9)T(5), (AC)(2)(AT)(8)T(5), (AC)(2)(AT)(7)T(7), (AC)(3)(AT)(7)T(5), (AC)(2)(AT)(8)T(9), (AC)(3)(AT)(8)T(5), (AC)(2)(AT)(10)T(3), (AC)(2)(AT)(11)T(3), and (AC)(2)(AT)(7)T(5) at position -530. Frequencies of 9 (AC)(n)(AT)(x)T(y) polymorphisms were 33.2%, 29.1%, 24.1%, 5.4%, 3.2%, 1.8%, 1.4%, 0.9%, and 0.9% respectively. It is concluded that rich (AC)(n)(AT)(x)T(y) polymorphisms at the -530 bp region in the BP1 binding site upstream of ß-globin gene are found in Chinese Han population. (AC)(2)(AT)(9)T(5), (AC)(2)(AT)(8)T(5), and (AC)(2)(AT)(7)T(7) are 3 major polymorphisms among Chinese Han population, and (AC)(3)(AT)(8)T(5) is a novel polymorphism at the -530 bp region. More studies should be done to explore relation between (AC)(n)(AT)(x)T(y) polymorphisms and ß-globin expression.

[Analysis of GJB2 gene coding sequence in patients with nonsyndromic hearing loss].

OBJECTIVE: To analyze the coding sequence of GJB2 gene in six pedigrees with nonsyndromic hearing loss in order to find deafness-causing mutations in the GJB2 gene, and to explore the inherent pattern of deafness-causing mutations in the GJB2 gene. METHODS: Genomic DNA was extracted from peripheral blood for the probands and their family members. Coding sequence of the GJB2 gene was amplified by polymerase chain reaction, sequence variations were determined by DNA sequencing. Amplified fragments with overlapping peaks on sequencing chromatogram were sequenced by TA cloning in order to determine whether the mutations originated from the same allele. RESULTS: Mutations in the GJB2 gene were found in 4 out of the 6 pedigrees with nonsyndromic hearing loss. Four types of mutations were detected in the GJB2 gene, which were 235delC, 299-300delAT, 79G>A+341A>G, and 109G>A. Compound heterozygous polymorphisms 79G>A and 341A>G, and mutations 109G>A and 235delC had deafness-causing effects. CONCLUSION: Heterogeneous mutations of the GJB2 gene are frequently seen in patients with nonsyndromic hearing loss. Sometimes, polymorphisms may cause deafness when they are combined. Environmental factors and other genes may contribute to hearing loss caused by the GJB2 gene mutations.

[Single nucleotide polymorphisms of ß-globin gene in ß-thalassaemia patients].

This study was aimed to analyze the ß-globin gene sequence and single nucleotide polymorphisms of the ß-globin gene in ß-thalassaemia patients from Shenzhen area, and to explore linkage relationships between ß-globin gene mutations and single nucleotide polymorphisms. Genomic DNA was extracted from peripheral leukocytes in 125 ß-thalassaemia patients from Shenzhen population. ß-globin gene was amplified by polymerase chain reaction, mutations and single nucleotide polymorphisms in the ß-globin gene were determined by DNA sequencing. The results indicated 10 types of mutation and 12 single nucleotide polymorphism loci were found in the ß-globin gene of 114 ß-thalassaemia patients. Linkage disequilibrium between mutations and single nucleotide polymorphisms was found in 6 loci including 6 haplotypes among these single nucleotide polymorphism loci in the ß-globin gene. It is concluded that a number of single nucleotide polymorphisms is found in the ß-globin gene, where an average of one single nucleotide polymorphism every 230 bp there is. Linkage disequilibrium occurs between ß-thalassaemia mutations and some haplotypes in single nucleotide polymorphism loci. This study may be helpful to gene diagnosis for ß-thalassaemia patients.

A novel androgen receptor gene mutation in a Chinese patient with complete androgen insensitivity syndrome.

OBJECTIVE: To identify the underlying androgen receptor gene mutation in a Chinese patient with typical symptoms of complete androgen insensitivity syndrome. STUDY DESIGN: A Chinese female phenotype with 46, XY karyotype was diagnosed because of primary amenorrhea. Mutations were determined by polymerase chain reaction followed by DNA sequencing. RESULTS: DNA sequencing of the androgen receptor gene showed a G2439T transition causing E442X mutation in exon 1 in the patient with complete androgen insensitivity syndrome. The E442X mutation was a new de novo non-sense mutation in exon 1 of the androgen receptor gene. This non-sense mutation is located in the N-terminal transactivation domain and leads to a predicted truncated protein of 441 amino acids with loss of the end part of the N-terminal transactivation domain, and the DNA-binding and ligand-binding domain. CONCLUSION: This E442X non-sense point mutation has not been described previously in cases of androgen insensitivity syndrome, and could lead to the synthesis of a short truncated non-functional androgen receptor probably responsible for the phenotype of complete androgen insensitivity syndrome in the affected individual. Gonadectomy should be planned to eliminate the risk of gonadal malignancy.