Construction of antisense RNA expression vectors and correction of splicing defect in human β-globin gene (IVS-2-654 C→T mutant) in HeLa cells

The antisense fragments, which were available inin vitro system, were cloned into the mammalian expression vector pcDNA3, and were transfected into H654 cells, a mammalian cell line stably expressing the thalassaemic (IVS-2-654 C→T) human β-globin gene. In these transfected cells, the level of correctly spliced β-globin mRNA in total β-globin mRNA (β/(β + β*)) was improved from 0.07 (0 d) to 0.22 (3 d), and this effect persisted for up to 15 d post transfection. All the results demonstrated that antisense RNAs were able to be transcribed from the antisense fragment expression vectors stably and effectively suppressed aberrant splicing pattern of the mutated β-globin gene (IVS-2-654 C→T) and restored correct splicing pathway. This work provided a novel approach with potential clinical significance to gene therapy of this kind of splicing mutants including β-thalassaemia (IVS-2-654 C→T) by antisense RNAs.     

Impaired bone formation and osteopenia in heterozygous β<Superscript>IVSII-654</Superscript> knockin thalassemic mice

β-thalassemia caused by the C→T mutation at nucleotide 654 of the intron 2 (β^IVSII-654) results in aberrant splicing of β-globin RNA, leading to an almost absence of β-globin synthesis. Although trabecular and cortical bone loss was previously reported in β-thalassemic mice with deletion of β-globin gene, the microscopic changes in trabecular structure in β^IVSII-654 thalassemic mice remained elusive. Here, we investigated the macroscopic and microscopic bone changes in 12-week-old β^IVSII-654 knockin thalassemic mice by dual-energy X-ray absorptiometry (DXA) and histomorphometric analysis, respectively. DXA revealed a decrease in bone mineral density in the lumbar vertebrae and tibial metaphysis, but not in the femoral diaphysis, suggesting that β^IVSII-654 thalassemia predominantly led to osteopenia at the trabecular site, but not the cortical site. Further histomorphometric analysis of the tibial secondary spongiosa showed that trabecular bone volume was significantly decreased with the expansion of marrow cavity. Decreases in osteoblast surface, osteoid surface, mineral apposition rate, mineralizing surface, and mineralized volume were also observed. Moreover, trabecular bone resorption was markedly enhanced as indicated by increases in the osteoclast surface and eroded surface. It could be concluded that β^IVSII-654 thalassemia impaired bone formation and enhanced bone resorption, thereby leading to osteopenia especially at the trabecular sites, such as the tibial metaphysis.     

Development of a PCR/Ligase Detection Reaction/Nanogold-Based Universal Array Approach for the Detection of Low-Abundant DNA Point Mutations

The aim of this study was to investigate the feasibility of combining PCR and ligase detection reaction (LDR) with a novel nano-gold-based universal array for the detection of low abundance point mutations from fetal DNA in maternal plasma samples. The sequence with the target point mutation was first amplified by PCR and then used as a template for LDR in which the upstream specific primer contains a tag sequence at the 5′-end. After hybridization to the probes of a universal array containing anti-tag sequences, the ligated products were bound to streptavidin-labeled nano-gold particles and the hybridization signals were amplified by silver staining. The PCR/LDR/universal array was first tested for sensitivity with nano-gold-based detection, and then this system was applied to detect the low abundance specific mutation IVS2 654(C→T) of the β-globin gene in a model using maternal plasma samples. The nano-gold-based method unambiguously identified a single mutation at a sensitivity of 1:1000. This approach was applied to detect the paternally inherited IVS2 654(C→T) mutation from thirty maternal plasma samples. The results were consistent with those obtained by PCR/reverse dot blot of amniotic fluid cell DNA. The PCR/LDR/nano-gold-based universal array is able to detect low-abundance point mutations with high sensitivity.     

The Cretan type of non-deletional hereditary persistence of fetal hemoglobin [Aγ–158C→T] results from two independent gene conversion events

We report a new type of non-deletional hereditary persistence of fetal hemoglobin that is due to a C→T transition at position –158, relative to the Cap site of the human Aγ-globin gene. This mutation was identified in three unrelated adult cases presenting slightly elevated levels of fetal hemoglobin (Hb F), i.e. 2.9–5.1%, and normal hematological indices. Our sequencing results, from both polymerase chain reaction-amplified and subcloned DNA fragments, indicate that the Aγ–158C→T mutation occurred by two independent gene conversion events in the three cases studied. In addition, hematological and molecular data, including restriction fragment length polymorphism haplotyping in the β-globin gene cluster, extended haplotype analysis inside the γ-globin gene region and routine analysis of three tandem repeat loci (D1S80, 3′-HVR/apoB and F8vWf), led us to conclude that the Aγ–158C→T mutation in one of the three cases occurred recently in the parental germ line (P=99.47%), representing the first example of a de novo gene conversion event identified in humans. Received: 10 November 1997 / Accepted: 10 February 1998     

可随HeLa细胞传代而传递的反义RNA真核表达系统

为研究反义ＲＮＡ表达载体在细胞内的稳定性,构建了一个特异性针对β地中海贫血基因ＩＶＳ－２－６５４Ｃ→Ｔ（β６５４）突变ｍＲＮＡ前体异常剪接位点的反义ＲＮＡ表达载体ｐＣＭＶＡ．ｐＣＭＶＡ转染β６５４ＨｅＬａ细胞后,通过ＲＮＡ定量检测反义片段对β６５４ｍＲＮＡ异常剪接的纠正作用;再从转染后传代５次并冰冻保存１年的ＨｅＬａ细胞中回收反义表达载体,转染另外的β６５４ＨｅＬａ细胞,同样检测它对β６５４ｍＲＮＡ异常剪接的纠正作用．结果显示该载体在细胞传代前后均能阻断β６５４异常剪接,部分恢复其正常剪接途径：用回收的ｐＣＭＶＡ转染β６５４ＨｅＬａ细胞后,正常剪接的βｍＲＮＡ水平［β／（β＋β＊）］由０．０５上升到处理后１５ｄ的０．４８,而２种对照质粒处理后对这一比值影响不大．表明ｐＣＭＶＡ可在ＨｅＬａ细胞中随着细胞传代而传递下去,并保持结构与功能完整     

β-Thalassemia and βA globin gene haplotypes in Mexican mestizos

β-globin haplotypes of 20 β-thalassemia (β-thal) and 87 β^A Mexican mestizo chromosomes were analyzed to ascertain the origin of the β-thal alleles and the frequencies and distribution of the β^A haplotypes among northwestern Mexican mestizos. Sixteen β-thal chromosomes carried six Mediterranean alleles [five codon 39 C→T; two IVS1:1 G→A; two IVS1:5 G→A; three IVS1:110 G(A; one codon 11 (–T) and three (δβ)°-thal]; the remaining four were linked to three rare alleles (two –28 A→C and one each: –87 C→T and initiation codon ATG→GTG). Among the 87 β^A chromosomes, 17 different 5′ haplotypes with frequencies for 1, 3, 2 and 5 of 39.0%, 17. 2%, 9.2% and 6.9%, respectively, were observed. The β-haplotype analysis showed that 13 out of 16 Mediterranean chromosomes could easily be explained by gene migration; however, one codon 39 associated with haplotype 4 (– – – – + + –), one IVS1:1 with haplotype 1(+ – – – – + +) and one IVS1:5 G→A, may represent separate mutational events. Analysis of the rare alleles showed that the –28 A→C mutation was associated with the commonest β^A haplotype in Mexican mestizos, Mediterraneans and the total world population; therefore an independent origin cannot be ruled out. The –87 C→T and initiation codon ATG→GTG were found with β-haplotypes different from the reported ones, suggesting an indigenous origin. Received: 23 April 1996 / Revised: 10 September 1996     

Hb Costa Rica or · 2 ‚ 277(EF1)His→Arg: the first example of a somatic cell mutation in a globin gene

We have identified a minor hemoglobin component (∼5%) in the blood of a healthy Costa Rican female, but not in her mother and two brothers (father not studied), that has an His→Arg replacement at position β77 (Hb Costa Rica). No other amino acid replacements were observed and no β- or γ-chain-like peptides were present. Hb Costa Rica has a normal stability. Sequence analyses of numerous polymerase chain reaction (PCR)-amplified segments of DNA that contain exon 2 of the β gene failed to identify a CAC→CGC (His→Arg) mutation. The same was the case when cDNA was sequenced, indicating that a β-Costa Rica-mRNA could not be detected with this procedure. Gene mapping of genomic DNA with BglII, BamHI, and HindIII gave normal fragments only and with the same intensity as observed for the fragments of a normal control. The quantities of the β chain variants Hb J-Iran and Hb Fukuyama with related mutations at β77 vary between 30% and 45% in heterozygotes, whereas that of Hb F-Kennestone with the same His→Arg mutation but in the ^Gγ-globin gene, is a high 40%–45% (as percentage of total ^Gγ) in a heterozygous newborn. These different observations exclude a heterozygosity of the A→G mutation at codon β77, as well as a deletion comparable to that of Hbs Lepore or Kenya, or a β-globin gene duplication, and point to a nontraditional inheritance of Hb Costa Rica. Allele-specific amplification of cDNA with appropriate primers identified the presence of a low level of mutated mRNA in the reticulocytes of the patient, which was confirmed by dotblot analysis of the same material with ³²P-labeled probes. Comparable amplification products were not observed in genomic DNA. The A→G mutation apparently occurred in a somatic cell at a relatively early stage in the development of the hematopoietic cell system, and Hb Costa Rica accumulated through rapid cell divisions in patchy areas in the bone marrow (somatic mosaicism). An unequal distribution of Hb Costa Rica over the red cells supports this possibility. Received: 25 August 1995 / Revised: 13 December 1995     

Platypus globin genes and flanking loci suggest a new insertional model for beta-globin evolution in birds and mammals

Background  

Vertebrate alpha (α)- and beta (β)-globin gene families exemplify the way in which genomes evolve to produce functional complexity. From tandem duplication of a single globin locus, the α- and β-globin clusters expanded, and then were separated onto different chromosomes. The previous finding of a fossil β-globin gene (ω) in the marsupial α-cluster, however, suggested that duplication of the α-β cluster onto two chromosomes, followed by lineage-specific gene loss and duplication, produced paralogous α- and β-globin clusters in birds and mammals. Here we analyse genomic data from an egg-laying monotreme mammal, the platypus (Ornithorhynchus anatinus), to explore haemoglobin evolution at the stem of the mammalian radiation.     

Uniformity in the nonsynonymous substitution rates of embryonic β-globin genes of several vertebrate species

Summary The nucleotide substitution rate in structural portions of the embryonic β-globin genes of placental mammals is lower than that for the adult β-globin genes. This difference occurs entirely within the class of substitutions that result in nonsynonymous (replacement) differences between these genes, and therefore represents a constraint on the structure of the mammalian embryonic β-globin proteins relative to the adult proteins (Shapiro et al. 1983; Hardison 1984). A similar effect has also been observed in marsupial mammals (Koop and Goodman 1988). In an effort to determine whether the observed rates are evidence of a uniform degree of selective constraint on the embryonic β-globin genes, analyses were performed that compared replacement substitution rates. The analyses reveal that embryonic β-globin genes appear to have been fixing replacement substitutions at nearly the same average rate not only in placental and marsupial mammals but in avian and amphibian species as well. In contrast, the adult β-globin genes from these organisms appear to have a more variable rate of replacement substitution with an especially low rate for birds. In the chicken (Gallus gallus), the adult β-globin gene replacement substitution rate appears to be lower than the embryonic replacement substitution rate.     

Beta-thalassaemia in indigenous Belgian families: identification of a novel mutation

The molecular basis of β-thalassemia was investigated at the DNA level in 28 Belgians from 14 unrelated families. All the patients were heterozygous for β-thalassaemia. Seven different mutations were identified using a combination of dot-blot hybridization with allele-specific oligonucleotide probes and direct automated fluorescence-based DNA sequencing. Among these mutations, four are commonly found in the Mediterraneans – codon 8 (–AA), IVS-I-1 (G→A), IVS-I-6 (T→C) and codon 39 (C→T) – and two have occasionally been reported – initiation codon (T→C) and codon 35 (C→A). The last mutation, a –CC deletion at codons 38/39, appears to be a novel mutation and can routinely be investigated by AvaII restriction on amplified DNA. We report our findings, discuss the diversity of the mutations found in Belgium and show the usefulness of direct DNA sequencing in a population in which the molecular defects of β-thalassaemia have yet to be characterized and in which screening is hampered by the wide range of potential mutations. Received: 8 December 1995 / Revised: 7 February 1996     

Detection of responsible mutations for beta thalassemia in the Kermanshah Province of Iran using PCR-based techniques

Beta Thalassemia has been reported to be a common genetic disorder in Iran. To establish the molecular spectrum of the beta thalassemias in the Kermanshah Province of Iran, 185 unrelated beta thalassemia patients with Kurdish ethnic background were studied (181 β-thalassemia major and 4 β-thalassemia intermedia). Using polymerase chain reaction-amplification refractory mutation system (PCR-ARMS), restriction fragment length polymorphism (RFLP) and direct genomic sequencing twenty different mutations were identified accounting for 98.1% of the alleles. Approximately 80.8% of β-thalassemia chromosomes had β⁰ mutation. The most prevalent mutation was the IVSII-1 (G→A) (32.97%), followed by CD8/9 +G (13.51%), IVSI-110 (C→T) (8.38%), CD 36/37 −T (7.84%), CD8 −AA (5.94%), CD15 (G→A) (4.86%) and IVSI-1 (G→A) (4.59%). All of these mutations accounted for 78.1% of the alleles. The results described here will be of valuable help in the development of successful prevention programs for the population of Kermanshah.     

β-Thalassemia major resulting from a compound heterozygosity for the β-globin gene mutation: further evidence for multiple origin and migration of the thalassemia gene

Summary We describe in a Japanese family -thalassemia resulting from a compound heterozygosity for a -globin gene mutation. One mutation is a C-to-T transition at IVS-2 nucleotide position 654 on the background of Mediterranean haplotype IX. Another mutation is a G-to-A transition at IVS-2 nucleotide position 1, associated with a novel haplotype XL The occurrence of these mutations on various chromosomal backgrounds provides strong evidence for an interplay of gene migration, interallelic gene conversion, and multiple origins of the same mutation.     

Towards β-globin gene-targeting with integrase-defective lentiviral vectors

We have developed an integrase-defective lentiviral (LV) vector in combination with a gene-targeting approach for gene therapy of β-thalassemia. The β-globin gene-targeting construct has two homologous stems including sequence upstream and downstream of the β-globin gene, a β-globin gene positioned between hygromycin and neomycin resistant genes and a herpes simplex virus type 1 thymidine kinase (HSVtk) suicide gene. Utilization of integrase-defective LV as a vector for the β-globin gene increased the number of selected clones relative to non-viral methods. This method represents an important step toward the ultimate goal of a clinical gene therapy for β-thalassemia.     

−509C>T polymorphism in the TGF-β1 gene promoter,impact on the hepatocellular carcinoma risk in Chinese patients with chronic hepatitis B virus infection

Hepatocellular carcinoma (HCC) is one of the most common malignant tumors worldwide. The risk for developing HCC increases with severity of inflammation and fibrosis. Transforming growth factor-β1 (TGF-β1) is most frequently upregulated in tumor cells. The most studied −509C>T polymorphism of TGF-β1 gene has been associated with colorectal, gynecologic, and lung cancers. To assess whether this polymorphism in TGF-β1 gene is associated with susceptibility to and/or clinicopathologic characteristics of HBV-related HCC, a total of 575 patients with chronic HBV infection and 299 healthy volunteers with no evidence of recent or remote HBV infection were prospectively enrolled. The patients were divided into two groups: those without (n = 196) and those with HCC (n = 379). These 379 HCC patients with chronic HBV infection were designated as cases, the remaining 196 patients without HCC and 299 healthy volunteers served as disease and healthy controls, respectively. −509C>T polymorphism in the TGF-β1 gene promoter was studied using restriction fragment-length polymorphism. In addition, tumor tissues of liver (n = 60) were obtained from the studied HCC patients for measurement of TGF-β1 mRNA expression levels. We also assessed the plasma TGF-β1 levels of HBV patients without (n = 94) or with HCC (n = 136) and healthy subjects (n = 120). In our study group, the risk of HCC in Chinese patients with HBV infection was significantly lower with the TT genotypes than in those with the CC genotypes at position −509 of TGF-β1 gene (P = 0.01). In addition, in the case group, patients with the CC genotype had a statistically significant higher median plasma TGF-β1 or liver tumor tissue TGF-β1 mRNA level compared with the individuals with the TT genotype. However, in a subsequent analysis of the association between this polymorphism and clinicopathological characteristics including tumor number, size, grade, stage, and invasiveness, there was no significant difference in both the distribution of genotype or allelic frequency within HCC patients, indicating that −509C>T exchange in TGF-β1 gene may play an important role in the occurrence, not the progression of HBV-related HCC through influencing plasma concentrations of TGF-β1 or TGF-β1 mRNA expression of liver tumor tissue.     

Mutations in the exon 10 of prolactin receptor gene change the egg production performance in Wanjiang white goose

To select the molecular genetic markers related to egg performance of Wanjiang white goose, prolactin receptor gene (PRLR) was adopted to be a candidate gene in our study. Five pairs of primers (P1–P5) were designed to detect the SNPs of PRLR gene by PCR-SSCP method. The results revealed that polymorphisms were discovered in the PCR products amplified with P4 primers in PRLR exon 10, three genotypes were found: AA, AB and AC. The sequence of AB genotype is the same as original sequence (DQ660982) in NCBI. There are five mutations in AA genotype: C → A at 840 bp, C → T at 862 bp, T → C at 875 bp, T → A at 963 bp, A → T at 989 bp, resulting in amino acid mutations: His → Asn, Thr → Ile, Asn → Lys, Thr → Ser, and synonymous mutation at 875 bp. Sequencing revealed five mutations in AC genotype: G → T at 816 bp, A → T at 861 bp, C → T at 862 bp, T → C at 875 bp, A → G at 948 bp, causing amino acid mutations of Val → Phe, Thr → Phe, synonymous mutations at 875 and 963 bp. Besides, there are an N-glycosylation site (NQSR), three casein kinase II phosphorylation sites including SIIE, SKTE, and SLMD in AA genotype; three casein kinase II phosphorylation sites including SIIE, SKTE, and TLMD in AB genotype; three casein kinase II phosphorylation sites including SIFE, SKTE, and TLMD in AC genotype. The annual egg yielding of AB genotype geese are significantly more than those of AA and AC genotype geese on the average (P < 0.05). It is suggested for the first time that PRLR is a promising candidate gene that can affect egg performance in Wanjiang white goose.     

Restoration of ß-Globin Gene Expression in Mammalian Cells by Antisense Oligonucleotides That Modify the Aberrant Splicing Patierns of Thalassemic Pre-mRNAs

Abstract

Antisense 2′-methoxy-oligoribonucleotides targeted to aberrant splice sites in two thalassemic human ß-globin pre-mRNAs, IVS2-654 and IVS2-705, expressed in HeLa cells efficiently restore correct splicing with the use of Lipofectamine, Cellfectin and DMRIE-C, in effect reactivating a defective ß-globin gene.     

Construction of SH-EP1-α4β2-hAPP695 Cell Line and Effects of Nicotinic Agonists on β-amyloid in the Cells

(1) Nicotinic acetylcholine receptors in central nervous system are thought to be new targets for Alzheimer’s disease. However, the most involved nicotinic receptor subtype in Alzheimer’s disease is unclear. α4β2 receptor is the most widely spread subtype in brain, involving in several important aspects of cognitive and other functions. We constructed cell line by transfecting human amyloid precursor protein (695) gene into SH-EP1 cells which have been transfected with human nicotinic receptor α4 subunit and β2 subunit gene, to observe effects of α4β2 receptors activation on β-amyloid, expecting to provide a new cell line for drug screening and research purpose. (2) Liposome transfection was used to express human amyloid precursor protein (695) gene in SH-EP1-α4β2 cells. Function of the transfected α4β2 receptors was tested by patch clamp. Effects of nicotine and epibatidine (selective α4β2 nicotinic receptor agonist) on β-amyloid were detected by Western blot and ELISA. Effects of nicotine and epibatidine on amyloid precursor protein (695) mRNA level were measured using real-time PCR. (3) Human amyloid precursor protein (695) gene was stably expressed in SH-EP1-α4β2 cells; Nicotine (1 μM) and epibatidine (0.1 μM) decreased intracellular and secreted β-amyloid in the cells; and activation of α4β2 receptors did not affect amyloid precursor protein (695) mRNA level. (4) These results suggest that the constructed cell line, expressing both amyloid precursor protein (695) gene and human nicotinic receptor α4 subunit and β2 subunit gene, might be useful for screening specific nicotinic receptor agonists against Alzheimer’s disease. Alteration of Aβ level induced by activation of α4β2 nAChR in our study might occur at a post-translational level.     

Regional distribution of β-thalassemia mutations in India

We have characterized the mutations in 1050 carriers of the β-thalassemia gene and analyzed their regional distribution in India. The majority of β-thalassemia carriers were migrants from Pakistan and their pattern of mutations differed from the rest. The frequency of the 619-bp deletion was 33.3% among the migrants from Pakistan, 8–17% in the northern states, and less than 5% in the other states. Among non-migrant subjects, the predominant mutation was IVS-I-5 (G→C), varying from 85% in the southern states and 66–70% in the eastern states to 47–60% in the northern states. The mutation IVS-I-1 (G→T) was observed at high frequency among the migrants from Pakistan (26.2%), but with very low/ zero frequency in the other states. Mutations at codons 8/9 (+G) and codons 41/42 (–CTTT) were distributed in all regions of India with a frequency varying from 3% to 15%. Only eight of 12 published rare mutations were observed in subjects from different parts of India. Mutations of codon 5 (–CT) and codons 47/48 (+ATCT) were found exclusively in migrants from Pakistan, and mutation –88 (C→T) was detected only in subjects from Punjab, Haryana, and Uttar Pradesh. Using the amplification refractory mutation system technique, mutations were successfully identified in 98.2% of subjects. Overall, 91.8% of the subjects had one of the five commonest mutations [IVS-I-5 (G→C), 34.1%; 619-bp deletion, 21.0%; IVS-I-1 (G→T) 15.8%; codons 8/9 (+G), 12.1%, and codons 41/42 (–CTTT), 8.7%], 5.9% of the subjects had a less common mutation, while 1.8% of the carriers remained uncharacterized. The application of this knowledge has helped to successfully establish a program of genetic counselling and prenatal diagnosis of β-thalassemia in order to reduce the burden of this disease in India. Received: 15 October 1996 / Accepted: 18 February 1997