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.

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.

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.

[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.

[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.

[Different splice of the calpain 3 gene in human skeletal muscle tissue and white blood cells].

OBJECTIVE: To investigate the splice variants of the calpain 3 gene existing in human skeletal muscle tissue and white blood cells, and to explore the feasibility of gene diagnosis using CAPN3 mRNA extracted from peripheral leukocytes. METHODS: Total RNA was extracted from peripheral blood and skeletal muscle tissue in healthy individuals. CAPN3 cDNAs were determined by reverse transcriptase polymerase chain reaction and DNA sequencing. CAPN3 cDNAs from peripheral leukocytes were compared with sequences obtained from skeletal muscle tissue. RESULTS: RT-PCR and DNA sequencing showed that the CAPN3 cDNAs comprised 24 exons in human skeletal muscle tissue, while the number of exons was 23 in white blood cells. Exon 15 was spliced out in human white blood cells. CONCLUSION: Splice variants exist in human skeletal muscle tissue and white blood cells. Gene diagnosis may omit the mutations of exon 15 using mRNA extracted from peripheral leukocytes. These findings suggest that mutation analysis of the CAPN3 cDNA should use skeletal muscle tissue as materials instead of peripheral blood.

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.

[Chromosome aberration in a full-term neonate with low birth weight using microarray comparative genomic hybridization].

OBJECTIVE: To analyze the chromosome aberration in a full-term male neonate with low birth weight, and to explore the possible causes for growth retardation in intrauterine development for the neonate. METHODS: Genomic DNA was extracted from peripheral leukocytes of the neonate. Detection of genomic DNA copy number gain and loss was performed using microarray comparative genomic hybridization. Chromosome karyotype was obtained from cultured lymphocytes for the neonate and his parents in order to identify the origin of chromosome aberration. RESULTS: Gain of 10q25.2-->qter (22 Mb) was observed in the full-term neonate with low birth weight. In addition, one chromosomal region, 15q26.2-->qter (5 Mb) was lost. The karyotype of the neonate was 46, XY, -15, +der(15), t(10;15)(q25;q26)pat. CONCLUSION: The full-term neonate with low birth weight had a partial trisomy of 10q25.2-->qter with a partial monosomy of 15q26.2-->qter, both of them may contribute to the growth retardation in intrauterine development for the neonate case.

[Changes of serum creatine kinase levels in children with Duchenne muscular dystrophy].

OBJECTIVE: Duchenne muscular dystrophy (DMD) usually occurs prior to 3 years old. The value of serum creatine kinase changes with clinical progression and age in patients with DMD. This study aimed to investigate the regularity in the changes of serum creatine kinase activities in children with DMD. METHODS: Peripheral blood samples were obtained from 40 children with DMD (ranged from 3-14 years). Serum creatine kinase levels were assayed by kinetic UV test. RESULTS: Serum creatine kinase level in the 40 DMD patients (ranged from 2 595- 45 495 U/L) was remarkably higher than the reference value (35-174 U/L). The highest serum creatine kinase level (average: 27750-31173 U/L) was found in 3-5 years old patients. Afterwards, serum creatine kinase level decreased with clinical progression and age, with a yearly average rate of decline was 8.7%. CONCLUSIONS: Serum creatine kinase level reaches a peak between 3 and 5 years old and then reduces with increasing age in children with DMD. The characteristic changes of serum creatine kinase are suspected to reflect the rate of muscle decay.

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 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.

Polymorphism of the D4Z4 locus associated with facioscapulohumeral muscular dystrophy 1A in Shanghai population.

OBJECTIVE: To investigate the D4Z4 repeats on chromosome 4q35 in normal individuals in Shanghai and analysis the polymorphism of the D4Z4 locus. METHODS: The length of D4Z4 repeats on chromosome 4q35 in 191 normal individuals in Shanghai was determined by pulsed-field gel electrophoresis and Southern blotting after double digestion with Eco RI and Bln I. The number of short D4Z4 repeats was counted after partial digestion with Kpn I. RESULTS: Among 191 normal individuals in Shanghai, seventeen showed the size of D4Z4 fragments ranged from 22 to 34 kb, i.e. 8.9% of individuals had fewer numbers of D4Z4 repeats. Of these 17 individuals, sixteen showed the short D4Z4 fragment on chromosome 4q35, and one low D4Z4 fragment was correlated to 4q35--> 10q26 translocation. CONCLUSION: The frequency of individuals having fewer numbers of D4Z4 repeats on chromosome 4q35 in Shanghai population is higher than that in Caucasian population although the short D4Z4 fragment on chromosome 4q35 is associated with facioscapulohumeral muscular dystrophy. These findings suggest that other factors may also contribute to facioscapulohumeral muscular dystrophy.

[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.

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.

[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.

[Dysferlin deficiency: the cause of limb-girdle muscular dystrophy 2B and Miyoshi myopathy in a Chinese pedigree].

OBJECTIVE: To identify an inbred Chinese pedigree with autosomal recessive muscular dystrophy and analyze the molecular defects. METHODS: Linkage analysis was conducted using short tandem repeat(STR) markers from the regions associated with limb-girdle muscular dystrophy type 2A(LGMD2A) through 2H. Multi-Western blot was performed with anti-calpain-3, anti-dysferlin, anti-gamma-sarcoglycan, anti-alpha-sarcoglycan, and anti-dystrophin monoclonal antibodies. Mutation was determined by reverse transcriptase-polymerase chain reaction and sequencing. RESULTS: Two-point linkage analysis showed significant Lod scores with markers from chromosome 2p13, the highest two-point Lod scores were obtained with D2S337 (Z(max)=1.86 at theta=0). Multi-Western blot confirmed dysferlin deficiency of muscle specimen from the proband. Mutation analysis revealed a novel 6429delG mutation on exon 53 of the DYSF gene for the proband. CONCLUSION: The authors identified an inbred Chinese pedigree with Miyoshi myopathy caused by a 6429delG on the DYSF gene. This mutation is predicted to result in premature termination of translation.

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.

[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.