Molecular basis of β-thalassemia in Thailand: analysis of β-thalassemia mutations using the polymerase chain reaction

Summary -Thalassemia mutations in 71 chromosomes of Thai patients from the northeast, the middle and the south of the country were investigated using dot blot hybridization of PCR (polymerase chain reaction)-amplified DNA with allelespecific oligonucleotide probes. Eight different known molecular defects were detected, at different frequencies. There was an amber mutation in codon 17, a C-T transversion at position 654 of IVS-2, a frameshift mutation between codons 71 and 72, an A-G transition at nucleotide -28 within the TATA box (known as Chinese mutations), a G-T transversion at position 1 of IVS-1 (an Indian mutation), a 4bp deletion in codons 41/42 and a G-C transversion at position 5 of IVS-1 (described as both Chinese and Indian mutations) and a Thai original mutation, an ochre mutation in codon 35. Analysis of the three unknown alleles by DNA sequencing of the cloned DNA fragment amplified by PCR revealed an A-G substitution at the second position of the codon for amino acid 19 (AAC-AGC). The analytic approach used in the present study and the characteristic distribution of mutations in each region of Thailand will prove useful for setting up a prenatal diagnosis program.     

The molecular defect in a family with mild atypical osteogenesis imperfecta and extreme joint hypermobility: Exon skipping caused by an 11-bp deletion from an intron in one COL1A2 allele

Summary We have investigated a family with an autosomal dominantly inherited connective-tissue defect causing extreme joint hypermobility, premature osteoporosis and late-onset fractures. Analysis of collagenous proteins from affected individuals showed a deletion in some 2(I) chains. Peptide mapping localized this to the CB peptide 2CB4, which covers the N-terminal one-third of the protein chain. Polymerase chain reaction amplification and sequencing of cDNA derived from this region of the mRNA identified á heterozygous deletion of the 54 by comprising exon 9. Similar analysis of the genomic DNA revealed an 11-bp deletion from bp3 to bp13 of IVS-9. This disrupts the consensus 5 splice signal (GTAAGT) and leads to exon skipping. In a family study of 13 affected and unaffected family members using both heteroduplex formation and direct analysis for the deletion, all of the affected, but no unaffected individuals, were found to carry the deletion. This generated a positive Lod score of 2.6 with the Liped programme.     

Rapid detection of deletions in the Duchenne muscular dystrophy gene by PCR amplification of deletion-prone exon sequences

Summary Using the polymerase chain reaction (PCR) technique, we have screened the DNA of 42 patients with Duchenne or Becker muscular dystrophy for deletions within the DMD gene. Two regions within putative deletion hot spots of this gene were tested, and deletions were found in 16.6% of patients. The oligonucleotide primers employed in this study initiate the amplification of exon sequences and were used to test the suitability and reliability of PCR in deletion screening and prenatal diagnosis using various numbers of cycles and artificial contamination ratios. We compared our approach with both multiplex DNA amplification and Southern blot analysis. A comparative evaluation of currently available techniques is presented.     

A rapid and simple electrophoretic method for the detection of mutations involving small insertion or deletion: application to β-thalassemia

Summary The 1.8-kb -globin gene fragments of DNAs from individuals heterozygous for nine different -thalassemia mutations involving 1, 2, 3, 4, or 25 basepair (bp) insertions or deletions were amplified by the polymerase chain reaction (PCR). The PCR products were subjected to electrophoresis on aqueous 8% polyacrylamide gel. In each heterozygote with either a 2 to 25 bp deletion, but not with a 1 bp insertion, two slower migrating bands representing heteroduplexes in addition to the 1.8-kb homoduplex band were seen. The electrophoretic positions of these slower migrating bands were characteristic of each mutation studied. By co-amplification with known normal DNA, it was also possible to distinguish DNAs from normal individuals and from individuals who are homozygous for the small insertion/deletion mutations. These studies demonstrate that the heteroduplex formation generated in PCR can be applied as a simple method in the diagnosis of insertion/deletion mutations involving 2 to 25 bp in -thalassemias as well as in other genetic disorders.     

Noninvasive Prenatal Detection for Pathogenic CNVs: The Application in α-Thalassemia

Background

The discovery of cell free fetal DNA (cff-DNA) in maternal plasma has brought new insight for noninvasive prenatal diagnosis. Combining with the rapidly developed massively parallel sequencing technology, noninvasive prenatal detection of chromosome aneuploidy and single base variation has been successfully validated. However, few studies discussed the possibility of noninvasive pathogenic CNVs detection.

Methodology/Principal Findings

A novel algorithm for noninvasive prenatal detection of fetal pathogenic CNVs was firstly tested in 5 pairs of parents with heterozygote α-thalassemia of Southeast Asian (SEA) deletion using target region capture sequencing for maternal plasma. Capture probes were designed for α-globin (HBA) and β-globin (HBB) gene, as well as 4,525 SNPs selected from 22 automatic chromosomes. Mixed adaptors with 384 different barcodes were employed to construct maternal plasma DNA library for massively parallel sequencing. The signal of fetal CNVs was calculated using the relative copy ratio (RCR) of maternal plasma combined with the analysis of R-score and L-score by comparing with normal control. With mean of 101.93× maternal plasma sequencing depth for the target region, the RCR value combined with further R-score and L-score analysis showed a possible homozygous deletion in the HBA gene region for one fetus, heterozygous deletion for two fetus and normal for the other two fetus, which was consistent with that of invasive prenatal diagnosis.

Conclusions/Significance

Our study showed the feasibility to detect pathogenic CNVs using target region capture sequencing, which might greatly extend the scope of noninvasive prenatal diagnosis.     

Microdissection and microcloning of rye (Secale cereale L.) chromosome 1R

Chromosome 1R was microdissected and collected from mitotic metaphase spreads of rye (Secale cereale L.) by using glass needles. The isolated chromosomes were amplified in vitro by Sau3A linker adaptor-mediated polymerase chain reaction (PCR). After amplification, the presence of rye-specific DNA was verified by Southern hybridization. The second-round PCR products from five 1R chromosomes were cloned into a plasmid vector to create a chromosome-specific library, which produced approximately 220,000 recombinant clones. Characterization of the microclone library showed that the 172 clones evaluated ranged in size from 300–1800 bp with an average size of 950 bp, of which approximately 42% were medium/high copy and 58% were low/unique copy clones. Chromosome in situ hybridization confirmed that the PCR products from microdissected chromosomes originated from chromosome 1R, indicating that many chromosome 1R-specific sequences were present in the library. Received: 5 December 1998; in revised form: 15 April 1999 / Accepted: 29 April 1999     

Identification of a sex-specific SCAR marker in dioecious Pandanus fascicularis L. (Pandanaceae).

Pandanus fascicularis L. is a dioecious plant native to South Asia with significant numbers in coastal areas. With the aim of distinguishing male genotypes from female genotypes early in the vegetative growth phase, the current study was initiated using molecular markers. Based on the principle of bulked segregant analysis, the sampled plants were separated into 2 bulks depending on their sex. Of the 89 random amplified polymorphic DNA and inter-simple sequence repeat markers used, one decamer (OPO-08) consistently amplified a 1263 bp band in the males that was absent in the females. Its DNA sequence did not exhibit significant similarity to previously characterized sequences, but the presence of mononucleotide and dinucleotide repeats suggested that it was a repeat-rich region. A sequence-characterized amplified region marker (MSSRF-01) designed for this fragment continued to amplify the specific allele in all the male plants. Southern hybridization performed using the sex-specific fragment as a probe yielded results consistent with those previously obtained by polymerase chain reaction. These results strongly suggest that MSSRF-01 is a male-specific molecular marker. With no information available on the presence of sex chromosomes in Pandanus, this marker can be used to differentiate the sexes.     

六例性反转综合征患者的分子遗传学分析

对六例性反转综合征患者(3例XX男性)(3例XY女性)用Y-特异性DNA探针进行了Southern印迹杂交分析,并用PCR技术扩增了SRY基因部分序列。结果表明,1例XX男性缺乏源于Y染色体的杂交信号,也无SRY基因;其余2例XX男性和3例XY女性都检测到Yp-DNA序列和SRY基因。这对进一步阐明性反转综合征的病因和SRY基因的作用机制具有重要意义。     

Organization and evolution of alpha satellite DNA from human chromosome 11

The human alpha satellite repetitive DNA family is organized as distinct chromosomal subsets located at the centromeric regions of each human chromosome. Here, we describe a subset of the alpha satellite which is localized to human chromosome 11. The principal unit of repetition of this alpha satellite subset is an 850 bp XbaI fragment composed of five tandem diverged alphoid monomers, each 171 bp in length. The pentamer repeat units are themselves tandemly reiterated, present in 500 copies per chromosome 11. In filter hybridization experiments, the Alpha 11 probes are specific for the centromeric alpha satellite sequences of human chromosome 11. The complete nucleotide sequences of two independent copies of the XbaI pentamer reveal a pentameric configuration shared with the alphoid repeats of chromosomes 17 and X, consistent with the existence of an ancestral pentameric repeat common to the centromeric arrays of at least these three human chromosomes.     

Detection of 98% of DMD/BMD gene deletions by polymerase chain reaction

Summary We describe oligonucleotide primer sequences that can be used to amplify eight exons plus the muscle promoter of the dystrophin gene in a single multiplex polymerase chain reaction (PCR). When used in conjunction with an existing primer set, these two multiplex reactions detect about 98% of deletions in patients with Duchenne or Becker muscular dystrophy (DMD, BMD). Furthermore, these primers amplify most of the exons in the deletion prone hot spot region around exons 44 to 53, allowing determination of deletion endpoints and prediction of mutational effects on the translational reading frame. Thus, use of these PCR-based assays will allow deletion detection and prenatal diagnosis for most DMD/BMD patients in a fraction of the time required for Southern blot analysis.     

Alpha-thalassemia in the four major aboriginal groups in Taiwan

A total of 1309 unrelated blood samples from four major Taiwan aboriginal groups, including 522 of the Ami, 246 of the Bunun, 227 of the Atayal, and 214 of the Paiwan groups, were collected. Subjects with a mean corpuscular volume below 85 fl and Hb A2 values below 3.5% were further studied with Southern hybridization to determine the status of -globin genes. In the Ami, 43 (4.1%) chromosomes had -thalassemia 1 and 43 (4.1%) had -thalassemia 2. Of the 43 -thalassemia 1 chromosomes, 33 were of the Thailand, one of the Philippine, and nine of the Southeast Asian deletion. Of the 43 -thalassemia 2 chromosomes, 42 were of the type I rightward deletion and one was of leftward deletion. In the Bunun group, one chromosome (0.2%) was of the Thailand deletion and two (0.4%) were of type I rightward deletion. In the Atayal group, only one chromosome (0.2%) was of the Philippine deletion. In the Paiwan group, four chromosomes (0.9%) were of the Southeast Asian deletion and three (0.7%) were of the Thailand deletion. Among the four groups, the Ami had the highest prevalence of -thalassemia, which was also higher than that of the Chinese living in Taiwan.     

Chromosomal assignment and linkage analysis of the human glutathione S-transferase μ gene (GSTM1) using intron specific polymerase chain reaction

Oligonucleotide primers specific for intron 5 sequences were used to amplify a unique 718 bp fragment in the human GST gene. Using DNA from a panel of somatic cell hybrids it was possible to confirm the assignment of the GST1 locus to chromosome 1p and to refine localisation to 1p13 using Southern blot analysis of DNA from three-generation CEPH families and a GST specific DNA probe.     

Specific amplification of deleted mitochondrial DNA from a myopathic patient and analysis of deleted region with S1 nuclease

Heteroplasmy of the normal-sized and the deleted mitochondrial genome has been observed in mitochondrial myopathy. The deleted region of the genome in the skeletal muscle of a patient was analyzed both by the conventional Southern blot method and by the novel method of employing the combination of polymerase chain reaction and S1 nuclease digestion. The results obtained by these methods were compared. Southern hybridization using various mitochondrial DNA fragments localized the deletion from at least position 9020 to 14,955, but regions of uncertainty of 1 kb remained on both ends of the deletion. Using the polymerase chain reaction, a fragment from the deleted genome was specifically amplified by choosing a pair of primers surrounding the deletion, and two fragments adjacent to the starting and end of the deletion were amplified from the normal-sized genome. S1 nuclease analysis of the heteroduplexes formed among these fragments demonstrated that the deletion extended from positions 8650 +/- 50 to 15,660 +/- 60. This method does not require radioisotopes and, moreover, can determine the deleted region within 5 h, in contrast to the 2 days required by the conventional Southern blot analysis. These results indicate that the novel method is faster and more accurate than the conventional method for the determination of the deleted region of genome.     

Molecular and cytogenetic characterization of repetitive DNA sequences from Lolium and Festuca: applications in the analysis of Festulolium hybrids

Summary A set of species-specific repetitive DNA sequences was isolated from Lolium multiflorum and Festuca arundinacea. The degree of their species specificity as well as possible homologies among them were determined by dot-blot hybridization analysis. In order to understand the genomic organization of representative Lolium and Festuca-specific repetitive DNA sequences, we performed Southern blot hybridization and in situ hybridization to metaphase chromosomes.Southern blot hybridization analysis of eight different repetitive DNA sequences of L. multiflorum and one of F. arundinacea indicated either tandem and clustered arrangements of partially dispersed localization in their respective genomes. Some of these sequences, e.g. LMB3, showed a similar genomic organization in F. arundinacea and F. pratensis, but a slightly different organization and degree of redundancy in L. multiflorum. Clones sequences varied in size between 100 bp and 1.2 kb. Estimated copy number in the corresponding haploid genomes varied between 300 and 2×10⁴. Sequence analysis of the highly species-specific sequences from plasmids pLMH2 and pLMB4 (L. multiflorum specific) and from pFAH1 (F. arundinacea specific) revealed some internal repeats without higher order. No homologies between the sequences or to other repetitive sequences were observed. In situ hybridization with these latter sequences to metaphase chromosomes from L. multiflorum, F. arundinacea and from symmetric sexual Festulolium hybrid revealed their relatively even distribution in the corresponding genomes. The in situ hybridization thus also allowed a clearcut simple identification of parental chromosomes in the Festulolium hybrid.The potential use of these species-specific clones as hybridization probes in quantitative dot-blot analysis of the genomic make-up of Festulolium (sexual and somatic) hybrids is also demonstrated.Abbreviations bp Base pair (s) - CMA chromomycin A₃ - DAPI 4,6-diamidino-2-phenylindole - IPTG isopropyl -D-thio-galactopyranoside - kb kilobase pair(s) - NBT nitroblue tetrazolium chloride - X-gal 5-bromo-4-chloro-3-inonyl -D-galactopyranoside     

Molecular characterization of β-thalassemia mutations in Egypt

Summary The relative frequency of different -thalassemia mutations and their association with -globin haplotypes were studied in patients from the Nile delta region, Egypt, by means of the polymerase chain reaction, oligonucleotide hybridization and restriction analysis. We found that 8 mutations account for 77% of -thalassemia chromosomes in this population, the commonest being IVS-1 nt 110, IVS-1 nt 6 and IVS-1 nt 1. Each mutation was associated with a specific haplotype, with the exception of IVS-1 nt 110, found on 3 different chromosomal backgrounds. Our data show that testing for the 8 detectable mutations makes feasible prenatal diagnosis in 65% of at risk couples and exclusion testing in an additional 25% of cases.     

DNA-mediated transformation of a fungus employing a vector devoid of bacterial DNA sequences

Acremonium chrysogenum, a producer of cephalosporin C, was subjected to DNA-mediated transformaations using a vector without bacterial DNA sequences. Recombinant fungal strains were generated with a gel-purified DNA fragment, carrying only the mutated -tubulin gene from A. chrysogenum. The lack of any bacterial DNA was verified by Southern hybridization analysis and polymerase chain reaction amplifications to detect even residual DNA sequences. This procedure can be referred to as a self-cloning experiment for which less restricted working regulations are needed. Finally, the transfer of a synthetic hirudin gene by contransformation demonstrated that any DNA molecule can be introduced into the A. chrysogenum genome without bacterial marker genes. This seems to be highly relevant for biotechnical processes in which safe recombinant producer strains are required to satisfy governmental restrictions.     

细胞质雄性不育高粱叶绿体 ndh D 基因的序列变异

片段SAAU－02 700特异地扩增自7种具可育细胞质的高粱材料的总DNA,含有叶绿体psa C(88bp)和ndh D(192bp)基因的部分序列。该片段与Eco Ri HindⅢ酶切的总DNA,线粒体DNA和叶绿体DNA杂交,在总DNA中获得了0.74kb的杂交带,而在叶绿体中获得0.74kb和0．45kb两条杂交带。与线粒体DNA无杂交;与经Hae Ⅲ酶切的总DNA杂交,在不育系中获得4．9kb的杂交带,而保持系的杂交带为4．45kb。参考GenBank中高粱的近缘物种玉米叶绿体基因组的序列,构建了ndh D基因区的酶切位点图谱,借此分析得出高粱不育系的叶绿体ndh D基因序列已发生改变。这种变异与高粱细胞质雄性不育反生的关系正在探讨中。     

Species-specific DNA sequences for identification of somatic hybrids between Lycopersicon esculentum and Solanum acaule

Summary Species-specific highly repeated DNA sequences can be used to screen the progeny of protoplast fusions combining different species. Such probes are easy to clone and can be detected by fast methods, e.g., hybridization to total genomic DNA. Furthermore, due to their high copy number, hybridization signals are strong and represent more than one locus, unlike isozymes or resistance markers. After cloning and screening for species-specific DNA sequences we characterized the highly repeated DNA sequences of the solanaceous species Solanum acaule and Lycopersicon esculentum var. gilva. DNA sequencing and hy ridization revealed a prominent, tandemly arranged satellite DNA repeat of 162 bp in Lycopersicon esculentum and a different satellite repeat of 183 bp, also tandemly organized, in Solanum acaule. Each repeat is absent in the respective other species. Therefore, we have used these DNA repeats as markers to distinguish regenerated interspecific somatic hybrids from the respective fusion partners. These hybrids were clearly identified by Southern hybridization and dot-blot assays to the respective ³²P-labelled satellite DNA.     

βA and βthal DNA haplotypes in Sicily

Summary A close association between specific restriction fragment polymorphism patterns and specific mutations in Mediterranean people with thalassemia has been demonstrated by Kazazian et al. (1984). This finding is useful to characterize the number and types of mutations in each ethnic group for setting up prenatal diagnosis in the first trimester of pregnancy by the oligonucleotide technique. For this reason we studied 99 ^thal and 46 ^A chromosomes in the Sicilian population. We found seven different cleavage patterns, not considering two new haplotypes so far uncharacterized. Many of the patients (68.3%) were genetic compounds for different haplotypes while only 31.7% were haplotype homozygotes. They may still be thalassemia compound heterozygotes. These findings confirm the molecular basis of the heterogeneity of thalassemia in Sicily.     

Two classes of 5S rDNA unit arrays of the silver fir, Abies alba Mill.: structure, localization and evolution

The structure and organization of the 5S ribosomal DNA units of the silver fir, Abies alba Mill., as well as their position in the chromosome complement were investigated. PCR amplification of the gene and nontranscribed spacer region, sequence analysis and Southern hybridization, using a homologous probe, detected DNA sequences of approximately 550 bp and 700 bp. Sequence analysis of the spacers revealed that the difference in length between the sequences occurred in the middle spacer region as a result of the amplification of a 75-bp sequence of the short unit class, which is organized in four 54- to 68-bp tandem repeats in the long spacer unit. The 5S rDNA transcribed region is 120 bp long and shows high sequence similarity with other gymnosperm species. The comparative analysis of 5 and 3 flanking sequences of 5S rRNA genes of silver fir and other gymnosperms indicates that A. alba spacer units have the same rate of evolution and are more closely related to Larix and Pseudotsuga than to Pinus and Picea. Southern hybridization and fluorescence in situ hybridization of metaphase chromosomes of A. alba suggest that the short and long spacer units are organized as separate tandem arrays at two chromosomal loci on chromosomes V and XI.