The molecular basis of beta-thalassemia in Thailand: application to prenatal diagnosis.

To enable the prenatal diagnosis of beta-thalassemia by direct detection of the mutant beta-globin genes, we have determined the spectrum of mutations causing this disease in Thailand. The techniques employed included a combination of synthetic oligonucleotide probe hybridization, direct sequencing of genomic DNA enzymatically amplified by the polymerase chain reaction, and cloning and sequencing of the beta-globin genes. A total of 116 beta-thalassemia genes from 78 Hb E/beta-thalassemia patients and from 19 homozygous beta-thalassemia patients were analyzed, and the mutation was characterized in 112/116 (97%) of them. Eleven mutations were found, of which four (-CTTT in codon 41/42, AAG----TAG in codon 17, C----T in position 654 of the IVS-2 region, and A----G in position -28 upstream of the beta-globin gene) accounted for 83%; two previously undescribed mutations have been identified. The spectrum of beta-thalassemia mutations is similar to that reported among the Chinese. However, within the Thai population itself, patients with homozygous beta-thalassemia show a wider spread of mutations in comparison with the Hb E/beta-thalassemia group, in whom the frameshift 41/42 mutation predominates at a frequency of 62%. This difference in distribution may reflect the difference in ethnic origin of the two groups. Characterization of these mutations should aid the planning of a prenatal diagnosis program for beta-thalassemia in Thailand.     

Testing of G----A mutation in position 110 of a minor intron of beta-globin genes in patients with thalassemia in Azerbaijan

A point mutation G-A in the 110 position of the beta-globin gene small intron has been revealed by cloning and sequencing from the material of a homozygote beta-thalassemia patient in Azerbaijan. In the present study two allele-specific oligonucleotide probes for testing the mutation have been synthesized. Assessment frequency of the mutation among the beta-thalassemia patients in Azerbaijan has been performed with the use of the amplified beta-globin gene fragments obtained by using the thermostable DNA-polymerase from T. thermophilus with the subsequent dot-hybridization in gel of the amplified material with the oligonucleotide probes. The possibility to test the mutation by hybridization of the oligonucleotide probes with the donors and beta-thalassemia patients restricted genomic DNA has been analyzed. Only one of 50 thalassemia alleles of beta-globin genes under study has been shown to possess the mutation mentioned.     

Characterization of mutations in the factor VIII gene by direct sequencing of amplified genomic DNA

In order to search for mutations resulting in hemophilia A that are not detectable by restriction analysis, three regions of the factor VIII gene were chosen for direct sequence analysis. Short segments of genomic DNA of 127 unrelated patients with hemophilia A were amplified by polymerase chain reaction. A total of 136,017 nucleotides were sequenced, and four mutations leading to the disease were found: a frameshift at codon 360 due to deletion of two nucleotides (GA), a nonsense codon 1705 due to a C----T transition, and two missense codons at positions 1699 and 1708. The first missense mutation (A----T) results in a Tyr----Phe substitution at a putative von Willebrand factor binding site. The second results in an Arg----Cys substitution at a thrombin cleavage site. In addition, we identified three rare sequence variants: a silent C----T transition at codon 34 which does not result in an amino acid change, a G----C change at codon 345 (Val----Leu), and an A----G change at the third nucleotide of intron 14. Direct sequence analysis of amplified DNA is a powerful but labor-intensive method of identifying mutations in large genes such as the human factor VIII gene.     

The beta-globin gene in Sardinian delta beta 0-thalassemia carries a C----T nonsense mutation at codon 39.

Sardinian delta beta 0-thalassemia is an inherited syndrome characterized by the inactivity of the beta-globin gene and the persistent activity of the fetal gamma-globin genes, particularly the A gamma-globin gene. Previous mapping studies with restriction enzymes failed to show any abnormality in the non-alpha globin gene cluster. We have now examined the possibility that this syndrome might result from a single rather than two different defects. Restriction enzyme polymorphisms linked to the delta beta 0-thalassemic non-alpha globin fragments were defined providing the basis for cloning the delta beta 0-thalassemic beta-globin gene from the DNA of a heterozygous patient. This gene appears to carry a C----T single mutation causing the appearance of a stop codon at amino acid position 39 of the beta-globin gene. This mutation was previously reported in beta 0-thalassemic patients, in linkage with different haplotypes. We conclude that Sardinian delta beta 0-thalassemia is the result of two separate mutations, the former one (unknown) responsible for persistent expression of gamma-globin genes, the latter for beta 0-thalassemia.     

Mapping of the vaccinia virus DNA polymerase gene by marker rescue and cell-free translation of selected RNA

The previous demonstration that a phosphonoacetate (PAA)-resistant (PAAr) vaccinia virus mutant synthesized an altered DNA polymerase provided the key to mapping this gene. Marker rescue was performed in cells infected with wild-type PAA-sensitive (PAAs) vaccinia by transfecting with calcium phosphate-precipitated DNA from a PAAr mutant virus. Formation of PAAr recombinants was measured by plaque assay in the presence of PAA. Of the 12 HindIII fragments cloned in plasmid or cosmid vectors, only fragment E conferred the PAAr phenotype. Successive subcloning of the 15-kilobase HindIII fragment E localized the marker within a 7.5-kilobase BamHI-HindIII fragment and then within a 2.9-kilobase EcoRI fragment. When the latter was digested with ClaI, marker rescue was not detected, suggesting that the PAAr mutation mapped near a ClaI site. The sensitive ClaI site was identified by cloning partial ClaI-EcoRI fragments and testing them in the marker rescue assay. The location of the DNA polymerase gene, about 57 kilobases from the left end of the genome, was confirmed by cell-free translation of mRNA selected by hybridization to plasmids containing regions of PAAr vaccinia DNA active in marker rescue. A 100,000-dalton polypeptide that comigrated with authentic DNA polymerase was synthesized. Correspondence of the in vitro translation product with purified vaccinia DNA polymerase was established by peptide mapping.     

Molecular nature of beta-thalassemia in Tajikistan: a four base pair deletion in codons 41-42 of the beta-globin gene]

Thirty tajiks, whose relatives had beta-thalassemia traits (revealed in previous investigations by determination of the HbA-2 and HbF levels) were selected to screen beta-thalassemia mutations. DNA samples from each individual were subjected to the PCR (polymerase chain reaction) to amplify the 635 bp beta-globin gene fragment. One additional band was detected in three samples after the amplified fragment underwent electrophoresis in 2% agarose gel and the EtBr was stained, and two additional ones were revealed by 6% PAAGE and staining of the EtBr. All additional bands migrated more slowly than appropriate 635 bp fragment. It is supposed that additional bands are heteroduplexes formed from the wild type chains and mutated chains carrying a deletion or insertion. The 4 bp deletion of the 41-42 (-tctt) was detected after the direct sequencing of the amplified fragments. This mutation is common among Chinese but it was not revealed in the Middle Asia populations. The mutation can be easily screened using the PCR and electrophoresis in 2% agarose gel or PAAG of the amplified beta-globin gene fragments.     

Identification of five rare mutations including a novel frameshift mutation causing beta zero-thalassemia in Thai patients with beta zero-thalassemia/hemoglobin E disease.

6 out of 14 uncharacterized beta-thalassemia alleles from 187 Thai beta-thalassemia/HbE patients were identified by direct sequencing of DNA amplified by polymerase chain reaction. A novel mutation occurring from an insertion of adenosine in codon 95, which results in a shift of the reading frame with terminator at the new codon 101, was detected in one patient. In addition, two frameshift mutations not previously reported among the Thai population were also detected in 3 patients: one with a deletion of thymidine in codon 15 and two with an insertion of cytidine in codons 27/28. A frameshift mutation that occurred from a cytidine deletion in codon 41 was also found in one patient in this study. The remaining case was an amber mutation, GAG-TAG, in codon 43 in exon 2 of the beta-globin gene. These mutations bring the number of mutations known to be present in the Thai population to a total of 20, 15 of which were detected in beta-thalassemia/HbE patients.     

Hemoglobin K?ln: analysis of linkage relationships between the mutant gene and polymorphic restriction sites in the beta-globin gene cluster

Nuclear DNA has been analyzed by means of restriction endonuclease mapping procedure to identify chromosomes that carry mutant Hb K?ln beta-globin genes in a family with individuals heterozygous for this disease. Inherited DNA polymorphisms within the beta-globin gene cluster yielded a direct linkage of the Hb K?ln mutation to haplotype constellations that are diagnostic for further offspring.     

Resolution of a missense mutant in human genomic DNA by denaturing gradient gel electrophoresis and direct sequencing using in vitro DNA amplification: HPRT Munich.

The combination of denaturing gradient gel electrophoresis (DGGE) and in vitro DNA amplification has allowed us to (1) localize a DNA mutation to a given 100-bp region of the human genome and (2) rapidly sequence the DNA without cloning. DGGE showed that a mutation had occurred, but the technique revealed little about the nature or position of that mutation. The region of the genome containing the mutation was amplified by the polymerase chain-reaction technique, providing DNA of sufficient quality and quantity for direct sequencing. Amplification was performed with a 32P end-labeled primer that allowed direct Maxam-Gilbert sequencing of the amplified product without cloning. HPRTMunich was found to contain a single-base-pair substitution, a C-to-A transversion at base-pair position 397. We report the generation of a 169-bp, wild-type DNA probe that encompasses most of exon 3 of the human hypoxanthine guanine phosphoribosyltransferase (HPRT) gene and contains a low-temperature melting domain of approximately 100 bp. HPRTMunich, an HPRT mutant isolated from a patient with gout, has a single amino acid substitution; the corresponding DNA sequence alteration must lie within the low-temperature melting domain of exon 3. We report the separation of HPRTMunich from the wild-type sequence using DGGE. In addition to base-pair substitutions, DGGE is also sensitive to the methylation state of the molecule. The cDNA for HPRT was cloned into a vector and propagated in Escherichia coli dam+ and dam- strains; thus, methylated and unmethylated HPRT cDNA was obtained.(ABSTRACT TRUNCATED AT 250 WORDS)     

Human urate oxidase gene: cloning and partial sequence analysis reveal a stop codon within the fifth exon

Using the cDNA and selected genomic probes of rat urate oxidase, we have screened the human genomic library and isolated seven clones; one clone (clone 13) contained exonic regions which correspond to the exons 5, 6, and 7 of rat urate oxidase gene. The nucleotide sequence was determined for these three exons and exon/intron junctions, and compared with the sequence from the rat gene. A mutation resulting in a stop codon TGA was found in the fifth exon of the human urate oxidase gene. Sequence analysis of the polymerase chain reaction amplified DNA, corresponding to the fifth exon of urate oxidase from DNA samples from four different individuals, confirmed the same TGA stop codon in all. This single stop codon mutation and/or other mutation(s) in this gene may be responsible for the lack of urate oxidase activity in the human.     

The detection of beta-globin gene mutations in beta-thalassemia using oligonucleotide probes and amplified DNA

DNA amplification combined with the use of synthetic oligonucleotide probes has become an important tool in the identification of base substitutions. We report the use of this DNA amplification technique for the detection of mutations in beta-thalassemia. A series of oligonucleotide primers are synthesized which span the beta-globin gene; one primer is complementary to the coding strand and the other to the non-coding strand. The primers are chosen so that there is little homology with other DNA segments, especially the delta gene. Each set of primers spans an area of the gene between 100 and 300 bp, while the suspected mutation point is located between these two primers. With the use of such a primer set, the beta-globin gene region is amplified by denaturation, annealing and DNA synthesis. The amplification cycle is repeated 25-30 times, using the Klenow fragment of DNA polymerase I. The resulting amplified DNA is hybridized with normal and synthetic deoxynucleotide probes using a standard dot-blot method. We have designed a set of primers and experimental conditions which should prove useful to diagnostic centers for detection of numerous beta-thalassemia mutations.     

Characterization of a spontaneous mutation to a beta-thalassemia allele.

We have studied a nuclear family containing a single child with severe beta-thalassemia intermedia, a Greek-Cypriot mother with hematological findings of beta-thalassemia trait, and a Polish father who is hematologically normal. Since both the child and her father were heterozygous for a DNA polymorphism within the beta-globin gene, it was possible to clone and sequence the beta-globin gene identical by descent from both the child and her father. A nonsense mutation in codon 121 (GAA----TAA) was found in the beta-globin gene of the child, while the same gene from her father lacked this mutation and was normal. This mutation has not been previously observed among over 200 beta-thalassemia genes characterized in Caucasians. Since the mutation eliminates an EcoRI site in the beta-globin gene, we could show that the mutation is not present in genomic DNA of the father. To rule out germinal mosaicism, sperm DNA of the father was also digested with EcoRI, and the mutant EcoRI fragment was not observed under conditions that would detect the mutation if it were present in at least 2% of sperm cells. Routine HLA and blood group testing supported stated paternity. In addition, studies with 17 DNA probes that detect multiple allele polymorphisms increased the probability of stated paternity to at least 10(8):1. These data provide evidence that the G----T change in codon 121 of the beta-globin gene in the child is the result of a spontaneous mutation that occurred during spermatogenesis in a paternal germ cell.     

Troubleshooting in gene splicing by overlap extension

A step-wise method for cloning intron-containing genes from genomic DNA is described. The two exons of the human proinsulin gene were separately amplified in two steps using, in the first step, completely homologous primers. This reduces unwanted interactions between mismatched primers and a complex DNA template such as genomic DNA. The fragments were amplified in a second step polymerase chain reaction (PCR) using mismatched primers that incorporated additional bases complementary to the other exon, and these products were spliced together in a third step PCR.     

Identification of Hb Hamilton or beta 11(A8)Val----Ile gene by the polymerase chain reaction amplification technique.

Amplification of the beta-globin gene by the PCR technique, followed by the enzymatic digestion of the DNA fragment obtained, was used to easily identify the human beta-globin variant Hb Hamilton which is characterized by the valine to isoleucine substitution at position 11. The result revealed the predicted G to A transition at codon 11 which abolishes a MaeIII restriction site. This mutation, which is rather common among Sardinians, is at the level of one of the five CpG dinucleotides of the beta-globin gene.     

A nonsense mutation in the cytochrome P450 CYP2D6 gene identified in a Caucasian with an enzyme deficiency

A novel mutation that generates a stop codon in the third exon of the gene encoding the cytochrome P-450 CYP2D6 was identified in a Caucasian having a deficiency of the isozyme, by means of single strand conformation polymorphism analysis of DNA fragments amplified by the polymerase chain reaction, followed by selective sequencing.     

Identification of Hb D-Punjab gene: application of DNA amplification in the study of abnormal hemoglobins.

Hemoglobin D-Punjab (or D-Los Angeles) is a common variant worldwide. It is also the most frequent abnormal hemoglobin in Xinjiang Uygur Autonomous Region of China. A large survey of hemoglobinopathy, including 142,171 people and 21 national/ethnic groups, was carried out in Xinjiang and indicated Hb D-Punjab accounted for 55.6% of the total hemoglobin variants there. Here we describe a simple way--EcoRI mapping of the amplified beta-globin DNA sampling from dried blood spots on filter paper blotters--of identifying the Hb D-Punjab gene. The primers were designed and synthesized to emzymatically amplify a 144-bp fragment of beta-globin gene which included codons beta 121 (GAA) and 122 (TTC) representing an EcoRI recognition site. The Hb D-Punjab gene could be easily detected by EcoRI digestion of the amplified DNA sequence on agarose gel because of a single base change at codon 121. The analysis of amplified DNA sampling from dried blood provides a very useful method for population study of Hb D-Punjab and will be of significance for demonstration of the occurrence of the Hb D-Punjab gene and for understanding of the relations among various nationalities.     

降低mRNA差异显示技术假阳性率的一种方法

为了探讨降低mRNA差异显示技术假阳性率的方法 ,进一步提高此技术的可靠性 ,提取了手术切除肝癌及非癌肝组织成对标本的总RNA ,逆转录获得cDNA片段 ,以mRNA差异显示方法筛选差异表达基因 ,选取较明显的一条差异表达条带 ,行进一步PCR扩增 .分别对PCR产物及其经TA克隆后随机挑选的 6个单克隆质粒DNA进行序列分析 ,并通过GenBank BLAST数据库进行序列的同源性比较 ,以Northern杂交予以来源确认 .自 72 0余条扩增条带中共选出 2 8条差异条带 .序列分析及同源性比较表明 ,所选择条带的PCR产物为一可能的新基因片段 ;而随机选择的 6个TA克隆质粒DNA中 ,有 4个为同一已知基因片段 ,一个为另一已知基因片段 ,一个为一可能的新基因片段 .同源性比较表明 ,PCR产物直接测序所得序列与TA克隆质粒DNA的 6个片段不具同源性 .结果表明 ,mRNA差异显示条带可能由 1条以上分子量相似的片段构成 ,直接对PCR产物行序列分析并以其为探针进行Northern杂交 ,是导致出现假阳性片段的原因之一 .将PCR产物进行TA克隆 ,对单克隆质粒DNA进行序列分析并以其为探针进行Northern杂交 ,可能是解决此问题的一种较好方法 .     

Amplification and direct nucleotide sequencing of cDNA from the lysate of low numbers of diploid human cells

The polymerase chain reaction technique is widely employed to amplify short segments of genomic DNA to determine if a specific change has occurred. However, some investigators need to sequence the entire coding region of mammalian genes, e.g., cellular ras genes or the gene encoding hypoxanthine (guanine) phosphoribosyl transferase (HPRT), to determine what specific changes have occurred. To do so, they isolate RNA from large populations of cells, amplify cDNA from the gene of interest, subclone the product, and sequence two or more isolates to determine the common mutation. We have developed a method to simplify this procedure by copying mRNA of the hprt gene directly from the lysate of a clone of mutant diploid human fibroblasts (e.g., 100 cells). We amplified the first and second strand of the cDNA of the gene of interest 10(10)- to 10(11)-fold, obtained 5 to 10 micrograms of DNA in less than 10 h, and sequenced the coding region directly without the need for RNA extraction or DNA template purification. By our method cDNA can be amplified directly from the lysate of just one human cell, but to avoid detecting random changes introduced by the polymerase, we lysed approx. 200 cells from a clone, each containing the identical mutation, amplified the cDNA, and determined the consensus sequence by direct nucleotide sequencing.