广东地区β地中海贫血复合缺失型α地中海贫血双重杂合子检出率

目的 研究广东地区β地中海贫血（地贫）杂合子复合α地贫双重杂合金的检出率。方法 采用反向点杂交法（RDB）诊断β地贫杂合子500例并进一步采用Gap-PCR法进行α地贫1基因检测,用α珠蛋白基因探针Southern印迹杂交限制性核酸内切酶酶谱分析法对其中400例β地贫杂合子中有26例合并α地贫2,其中17例为右侧缺失（αα／－α^3.7),9例为左侧缺失（αα/-α^4.2)。结论 广东地区β地贫杂合子复合α地贫1的检出率为8．6％,复合α地贫2的检出率为6．5％,其中复合右侧缺失型α地贫2的检出率为4．2％,复合左侧缺失型α地贫2的检出率为2．2％。     

Compensatory increase in alpha 1-globin gene expression in individuals heterozygous for the alpha-thalassemia-2 deletion.

alpha-Globin is encoded by the two adjacent genes, alpha 1 and alpha 2. Although it is clearly established that both alpha-globin genes are expressed, their relative contributions to alpha-globin messenger RNA (mRNA) and protein synthesis are not fully defined. Furthermore, changes that may occur in alpha-globin gene activity secondarily to the loss of function of one or more of these genes (alpha-thalassemia [Thal]) have not been directly investigated. This study further defines the expression of the two human alpha-globin genes by determining the relative levels of alpha 1 and alpha 2 mRNA in the reticulocytes of normal individuals and in individuals heterozygous for the common 3.7-kilobase deletion within the alpha-globin gene cluster that removes the alpha 2-globin gene (the rightward type alpha-Thal-2 deletion). To quantitate accurately the ratio of the two alpha-globin mRNAs, we have modified a previously reported S1 nuclease assay to include the use of 32P end-labeled probes isolated from alpha 1- and alpha 2-globin complementary DNA recombinant plasmids. In individuals with a normal alpha-globin genotype (as determined by Southern blot analysis [alpha alpha/alpha alpha]), alpha 2-globin mRNA is present at an average 2.8-fold excess to alpha 1. In individuals heterozygous for the rightward type alpha-Thal-2 deletion (-alpha/alpha alpha) the alpha 2/alpha 1 mRNA ratio is 1:1. These results suggest that the loss of the alpha 2-globin gene in the alpha-Thal-2 deletion is associated with a 1.8-fold compensatory increase alpha 1-globin gene expression.     

Locus assignment of alpha-globin structural mutations by hybrid-selected translation.

The two human alpha-globin genes, alpha 1 and alpha 2 located 3.4 kilobases apart on chromosome 16, encode identical alpha-globin proteins. A mutation in either gene could result in a structural hemoglobinopathy. It has only recently become possible to assign an alpha-chain mutant to one of these two loci by using recombinant DNA technology. While definitive, this approach has necessitated the cloning and sequencing of the specific gene in question. We present an alternative approach which results in rapid and definitive assignment of an alpha-globin mutation to its encoding genetic locus. This approach uses the technique of hybrid-selected translation. Reticulocyte RNA from individuals with alpha-globin mutations can be fractionated into beta-, alpha 9 (total)-, alpha 1-, and alpha 2-globin mRNA by selective hybridization of each mRNA species to its respective complementary DNA (cDNA) immobilized on nitrocellulose paper. Each mRNA purified in this way can be translated in vitro, and the mRNA species (and hence gene locus) encoding the globin mutant can then be directly identified by gel analysis of the radiolabeled translation products. This procedure can be used to identify globin mutants as alpha or beta and to localize alpha-globin mutants to the alpha 1 or alpha 2 gene. We have used this technique to localize the two alpha-globin mutants, alpha 125Pro (Hb Quong Sze) and alpha 47HIS (Hb Hasharon), to the alpha 2 locus. This approach could potentially be expanded to serve as an alternative to peptide analysis for the initial characterization of all globin structural mutants.     

Molecular basis for nondeletion alpha-thalassemia in American blacks. Alpha 2(116GAG----UAG).

An American black woman was found to have the phenotype of moderately severe alpha-thalassemia normally associated with the loss of two to three alpha-globin genes despite an alpha-globin gene map that demonstrated the loss of only a single alpha-globin gene (-alpha/alpha alpha). Several individuals in her kindred with normal alpha-globin gene mapping studies (alpha alpha/alpha alpha) had mild alpha-thalassemia hematologic values consistent with the loss of one to two alpha-globin genes. These data suggested the presence of a nondeletion alpha-thalassemia defect in this family which segregates with the intact alpha alpha gene cluster. An abnormally migrating and highly unstable alpha-globin gene product was demonstrated by in vitro translation of the reticulocyte mRNA from the proposita and this mutant alpha-globin protein was mapped to the alpha 2-globin gene by hybrid-selected translation. The abnormal alpha 2-globin gene was cloned and sequenced. A single base mutation that results in a premature termination codon was identified at codon 116 (GAG----UAG). The defined alpha-globin genotype of the proposita (-alpha/alpha 116UAG alpha) and the positioning of this nonsense mutation at the alpha 2-globin gene locus are fully consistent with the observed alpha-thalassemia phenotype.     

Molecular assay of -alpha(3.7) and -alpha(4.2) deletions causing alpha-thalassemia by denaturing high-performance liquid chromatography

OBJECTIVES: alpha-Thalassemia, the most common single gene disorder in humans, is due to the absence of one (-alpha/alphaalpha) or both (--/alphaalpha) of the two functional alpha-globin genes (alpha1 and alpha2). The -alpha(3.7) and -alpha(4.2) single gene deletions are common in Southeast Asian populations. Southern blotting analysis and gap PCR assay are commonly used for the detection of such alpha-thalassemia genotypes. The two genes are located on chromosome 16, with high homology (>96%). DESIGN AND METHODS: Based on the sequence variation within the two Z boxes, a denaturing high-performance liquid chromatography (DHPLC)-based assay was developed for rapid genotyping of the -alpha(3.7) and -alpha(4.2) alleles. To demonstrate the utility of this approach, 40 DNA samples with known genotypes were analyzed, including -alpha(3.7)/alphaalpha (7 cases), -alpha(4.2)/alphaalpha (4 cases), -alpha(3.7)/--(SEA) (6 cases), -alpha(4.2)/--(SEA) (3 cases), and 20 unaffected subjects (alphaalpha/alphaalpha). RESULTS: We successfully distinguished all of the alpha-thalassemia genotypes through their characteristic chromatograms of alpha1 and alpha2 genes. The accuracy of this technique for our sample was 100% sensitivity and specificity. CONCLUSION: This novel and alternative DHPLC-based alpha-thalassemia genotype assay is easy, rapid, and highly accurate. This technique enables the diagnosis of silent alpha+ thalassemia and hemoglobin H disease for large scale population screening.     

An initiation codon mutation (AUG----GUG) of the human alpha 1-globin gene. Structural characterization and evidence for a mild thalassemic phenotype.

alpha-globin is encoded by two adjacent genes, alpha 1 and alpha 2. Recent evidence suggests that these genes are not equally expressed and that the alpha 2-globin gene encodes the majority of alpha-globin. This finding would predict that a thalassemic mutation of the alpha 2-globin gene would result in a more severe loss of alpha-chain synthesis than a similar mutation in the alpha 1-globin gene. In a previous study we described a nondeletion alpha-thalassemia defect in the alpha 2-globin gene resulting from an AUG----ACG initiation codon mutation. In the present study we describe a different initiation codon mutation, AUG----GUG, present in the alpha 1-globin gene. The alpha 1- and alpha 2-globin gene initiation codon mutations result in similarly lowered levels of encoded mRNA. Despite the similarity of these two mutations, the alpha 2 mutant results in a more severe loss of alpha-globin synthesis and a more severe clinical alpha-thalassemia phenotype than the corresponding alpha 1-globin gene mutation. This difference reflects the dominant role of alpha 2-globin gene in overall alpha-globin synthesis.     

应用长片段PCR技术检测α珠蛋白基因组织的异常

目的：应用长片段多聚酶链反应（ＰＣＲ）技术检测α珠蛋白基因组织的异常。方法：设计一组引物分别互补于α２珠蛋白基因上游和α１珠蛋白基因下游的特异序列，应用长片段ＰＣＲ技术对７例具有正常和异常α珠蛋白基因组织个体进行分析。结果：正常的α珠蛋白基因组织（αα）扩增片段的长度为６．４ｋｂ；右侧缺失型的α珠蛋白基因组织（α－３．７）为２．６ｋｂ；而东南亚缺失型的α珠蛋白基因组织（－－ＳＥＡ）则无任何扩增片段；右侧增多型的α珠蛋白基因组织（αααａｎｔｉ３．７）的扩增片段为１０．２ｋｂ。结论：长片段ＰＣＲ技术为α珠蛋白基因组织异常的快速检测提供了一个新的方法。     

STEC-EPEC oligonucleotide microarray: a new tool for typing genetic variants of the LEE pathogenicity island of human and animal Shiga toxin-producing Escherichia coli (STEC) and enteropathogenic E. coli (EPEC) strains

BACKGROUND: Shiga toxin-producing Escherichia coli (STEC) and enteropathogenic E. coli (EPEC) are important emerging pathogens that can cause a severe and sometimes fatal illness. Differentiation of eae, tir, espA, espD, and espB gene variants of the locus of enterocyte effacement (LEE) pathogenicity island represents an important tool for typing in routine diagnostics as well as in pathogenesis, epidemiologic, clonal, and immunologic studies. METHODS: Type-specific oligonucleotide microarrays and a PCR scheme were designed and constructed for the detection and typing of genetic variants of the LEE genes. Oligonucleotide probes were tested for their specificity against the corresponding type strain by microarray hybridization using fluorescent DNA, either PCR-amplified (single, multiplex, long-range), chromosomal, or amplified chromosomal DNA. RESULTS: The PCR scheme and the oligonucleotide microarray allowed us to distinguish 16 variants (alpha1, alpha2, beta1, beta2, gamma1, gamma2/theta, delta/kappa, epsilon, zeta, eta, iota, lambda, mu, nu, xi, omicron) of the eae gene, 4 variants (alpha1, beta1, gamma1, gamma2/theta) of the tir gene, 4 variants (alpha1, beta1, beta2, gamma1) of the espA gene, 3 variants (alpha1, beta1, gamma1) of the espB gene, and 3 variants (alpha1, beta1, gamma1) of the espD gene. We found a total of 12 different combinations of tir, espA, espB, and espD genes among the 25 typed strains. CONCLUSIONS: The PCR scheme and the oligonucleotide microarray described are effective tools to rapidly screen multiple virulence genes and their variants in E. coli strains isolated from human and animal infections. The results demonstrate the great genetic diversity among LEE genes of human and animal STEC and EPEC strains.     

Validation of a reverse-hybridization StripAssay for the simultaneous analysis of common alpha-thalassemia point mutations and deletions.

BACKGROUND: alpha-Thalassemia is a worldwide disease and considered to be a major public health problem in countries within the so-called thalassemia belt. The complex genetics of alpha-thalassemias requires diagnostic methods with the capacity to screen rapidly and accurately for common causative mutations. METHODS: We developed and validated a reverse-hybridization assay (Alpha-Globin StripAssay) for the rapid and simultaneous detection of 21 alpha-globin mutations: two single gene deletions (-alpha(3.7); -alpha(4.2)), five double gene deletions [--(MED); --(SEA); --(THAI); --(FIL); -(alpha)(20.5)], alpha alpha alpha(anti-3.7) gene triplication, two point mutations in the alpha1 gene (cd 14 G>A; Hb Adana) and 11 point mutations in the alpha2 gene (initiation cd T>C; cd 19 -G; IVS1 -5nt; cd 59 G>A; Hb Quong Sze; Hb Constant Spring; Hb Icaria; Hb Pakse; Hb Koya Dora; polyA-1; polyA-2). RESULTS: Reliable genotyping of recombinant mutant clones and reference DNA samples was achieved by means of two corresponding test strips presenting parallel arrays of allele-specific oligonucleotides. The entire procedure from blood sampling to the identification of mutations required less than 6 h, and hybridization/detection was manual or automated. The diagnostic potential of this Alpha-Globin StripAssay was carefully evaluated on 272 pre-typed samples in a multicenter validation study. In 96.14% of the cases, StripAssay typing was completely concordant with the reference methods. CONCLUSIONS: The Alpha-Globin StripAssay proved to be a fast, easy-to-perform and reliable screening method to identify >90% of alpha-globin mutations in endemic areas worldwide.     

alpha-Thalassemia caused by an unstable alpha-globin mutant

In a previous study, molecular cloning of the alpha-globin genes from a patient with nondeletion Hb-H disease (genotype--/alpha alpha) showed that a single nucleotide mutation (CTG to CCG) in one of the genes resulted in a leucine to proline substitution. This paper describes the approach we used to detect the abnormal alpha-globin chain. The chain was identified using a cell-free translation system. It turned over rapidly both in vitro and in vivo in the patient's reticulocytes. The unusual feature of this unstable alpha-globin is that the alpha-globin deficiency causes alpha-thalassemia. Simple heterozygotes for this lesion (alpha Pro alpha/alpha alpha) resemble alpha-thalassemia carriers and do not exhibit the hemolytic anemia usually associated with unstable hemoglobins.     

βIVS—Ⅱ—1 G→A复合三联α基因所致的韩国人中间型β地中？…

目的 研究中间型β地中海贫血的分子机制,为中间型β地中海贫血的基因诊断提供科学依据。方法 应用聚合酶链反应（ＰＣＲ）,Ｓｏｕｔｈｅｒｎ印迹杂交和双链ＤＮＡ循环测序等技术,对在韩国首次发现的中间型β地中海贫血家系的α和β珠蛋白基因进行分析。结果 基因分析表明,该家系属典型的中间型β地中海贫血,它的分子病因是β珠蛋白基因的第２内含子５‘端的第１个碱基Ｇ→Ａ（ＩＶＳ－Ⅱ－１Ｇ→Ａ）突变和三联α珠蛋白基因     

Real-time multiplex PCR assay for rapid detection and toxintyping of Clostridium perfringens toxin producing strains in feces of dairy cattle

Clostridium perfringens is an anaerobic, gram-positive, spore-forming bacterium associated with a wide variety of diseases in domestic animals and humans. We have developed dual-labeled fluorescence hybridization probe (TaqMan((R)))-based real-time multiplex PCR assay for detection of toxin genes alpha (cpa), beta (cpb), iota (ia), epsilon (etx), beta2 (cpb2) and enterotoxin (cpe) of C. perfringens directly from cattle feces. The assay was standardized using ATCC reference strains of C. perfringens producing alpha, beta, iota, epsilon and enterotoxin, respectively. The assay for detection of beta2 toxin gene was standardized using a field strain of C. perfringens producing beta2 toxin. The minimum detection limit for the real time PCR assay ranged from 5 to 70 pg of DNA for the six toxin genes. A total of 307 fecal samples collected from seven dairy herds in Pennsylvania were analyzed using the multiplex assay. The real-time PCR assay revealed that cpa, cpb, ia, etx, cpb2 and cpe were detected in 68 (28.2%), 6 (2.5%), 6 (2.5%), 4 (1.6%), 164 (68%) and 11 (4.5%) of 241 PCR positive samples, respectively. The findings of the study revealed that C. perfringens beta2 toxin producing strains were widely prevalent in lactating cows in Pennsylvania and they may play an important role in C. perfringens associated diarrheal diseases.     

Evaluation of relative promoter strength in primary hepatocytes using optimized lipofection

For most genetic deficiencies manifested in the liver, maximization of gene expression in hepatocytes will be an important factor in achieving successful gene therapy. A rapid, highly efficient, and nontoxic method for transfecting DNA into hepatocytes was used to compare directly promoter strengths of various cellular and viral promoters. Conditions are described here for transfecting 5-10% of primary hepatocytes using the positively charged liposomes, Lipofectin. Cells are not damaged by this method as they continue to transcribe genes controlled by liver specific promoters and can survive for over 2 weeks in culture. We find that the cytomegalovirus, SR alpha, and beta-actin promoters are more active than the SV40, RSV, RNA polymerase II, albumin, alpha 1-antitrypsin, or phosphoenolpyruvate carboxykinase promoters. A simple TK promoter and a TK promoter with the polyoma enhancer (MCI) were almost completely inactive. This information will be useful in the construction of vectors designed to express genes efficiently in primary hepatocytes for purposes of gene therapy, although the stability of expression from these promoters will need to be demonstrated in hepatocytes in vivo.     

An erythroid chaperone that facilitates folding of alpha-globin subunits for hemoglobin synthesis

Erythrocyte precursors produce abundant alpha- and beta-globin proteins, which assemble with each other to form hemoglobin A (HbA), the major blood oxygen carrier. alphaHb-stabilizing protein (AHSP) binds free alpha subunits reversibly to maintain their structure and limit their ability to generate reactive oxygen species. Accordingly, loss of AHSP aggravates the toxicity of excessive free alpha-globin caused by beta-globin gene disruption in mice. Surprisingly, we found that AHSP also has important functions when free alpha-globin is limited. Thus, compound mutants lacking both Ahsp and 1 of 4 alpha-globin genes (genotype Ahsp(-/-)alpha-globin*(alpha/alphaalpha)) exhibited more severe anemia and Hb instability than mice with either mutation alone. In vitro, recombinant AHSP promoted folding of newly translated alpha-globin, enhanced its refolding after denaturation, and facilitated its incorporation into HbA. Moreover, in erythroid precursors, newly formed free alpha-globin was destabilized by loss of AHSP. Therefore, in addition to its previously defined role in detoxification of excess alpha-globin, AHSP also acts as a molecular chaperone to stabilize nascent alpha-globin for HbA assembly. Our findings illustrate what we believe to be a novel adaptive mechanism by which a specialized cell coordinates high-level production of a multisubunit protein and protects against various synthetic imbalances.     

Real-time quantitative PCR for the measurement of MYCN amplification in human neuroblastoma with the TaqMan detection system.

BACKGROUND: Neuroblastoma is the most common extracranial malignant solid tumor in children under 5 years and is characterized by a wide clinical and biological heterogeneity, from spontaneously regressive forms to cancers with a rapid and fatal progression. MYCN oncogene amplification is considered the most important prognostic factor to evaluate survival and therapeutic choices in these patients. METHODS: Here we present a new assay for rapid and accurate measurement of MYCN amplification, based on real-time quantitative PCR with the TaqMan(TM) reaction. The degree of MYCN amplification was derived from the ratio of the MYCN oncogene and the single-copy reference gene, beta-actin. The absolute abundance of these two genes in tumor sample DNA was obtained by extrapolation on external calibration curves generated with reference DNA. RESULTS: We found a variable degree of MYCN amplification, from 2 to 29, in 26 of 49 (53%) neuroblastomas. These results were well correlated to those obtained with a competitive PCR assay in the same samples (r = 0. 987). MYCN amplification was associated mainly with advanced cancer stages, and the analysis of overall survival confirmed that the measurement of MYCN amplification is a predictor of patient outcome in neuroblastoma. Patients without MYCN amplification had a cumulative survival significantly higher than patients with low (<9; P = 0.02) and high (>/=9; P = 0.03) oncogene amplification. CONCLUSION: The assay is rapid and reproducible and does not require any post-PCR analytical procedure.     

The molecular genetic analysis of polycystic kidney disease]

Autosomal dominant polycystic kidney disease (ADPKD) is one of common single gene disorders. The development of molecular genetic techniques has shown that mutant PKD1 gene assigned to ADPKD was closely linked to alpha-globin on the short arm of chromosome 16. This location was established when genetic linkage was found between ADPKD and a highly polymorphic region at the 3' end of the alpha-globin cluster (3' HVR). The discover of genetic linkage markers such as 3' HVR probe has provided a diagnostic test in presymptomatic stage. We performed this diagnostic test using DNA probes in 3 patients with ADPKD of one Japanese family. They also showed PKD1 gene linkage as previously described by Reeders et al. Linkage analysis of the PKD1 gene might be available to diagnostic test of ADPKD. DNA diagnosis of ADPKD however has to be performed carefully because of an ethical standpoint.     

Effects of alpha-thalassemia and sickle polymerization tendency on the urine-concentrating defect of individuals with sickle cell trait.

A defect in urine concentrating ability occurs in individuals with sickle cell trait (HbAS). This may result from intracellular polymerization of sickle hemoglobin (HbS) in erythrocytes, leading to microvascular occlusion, in the vasa recta of the renal medulla. To test the hypothesis that the severity of the concentrating defect is related to the percentage of sickle hemoglobin present in erythrocytes, urinary concentrating ability was examined after overnight water deprivation, and intranasal desmopressin acetate (dDAVP) in 27 individuals with HbAS. The HbAS individuals were separated into those who had a normal alpha-globin genotype (alpha alpha/alpha alpha), and those who were either heterozygous (-alpha/alpha alpha) or homozygous (-alpha/-alpha) for gene-deletion alpha-thalassemia, because alpha-thalassemia modulates the HbS concentration in HbAS. The urinary concentrating ability was less in the alpha alpha/alpha alpha genotype than in the -alpha/alpha alpha or -alpha/-alpha genotypes (P less than 0.05). After dDAVP, the urine osmolality was greater in patients with the -alpha/-alpha genotype than with the -alpha/alpha alpha genotype (882 +/- 37 vs. 672 +/- 38 mOsm/kg H2O) (P less than 0.05); patients with the -alpha/alpha alpha genotype had greater concentrating ability than individuals with a normal alpha-globin gene arrangement. There was an inverse linear correlation between urinary osmolality after dDAVP and the percentage HbS in all patients studied (r = -0.654; P less than 0.05). A linear correlation also existed for urine concentrating ability and the calculated polymerization tendencies for an oxygen saturation of 0.4 and O (r = -0.62 and 0.69, respectively). We conclude that the severity of hyposthenuria in HbAS is heterogeneous. It is determined by the amount of HbS polymer, that in turn is dependent upon the percentage HbS, which is itself related to the alpha-globin genotype.     

Multiplex PCR detection of GSTM1, GSTT1, and GSTP1 gene variants: simultaneously detecting GSTM1 and GSTT1 gene copy number and the allelic status of the GSTP1 Ile105Val genetic variant

The glutathione S-transferase (GST) genes GSTM1, GSTT1, and GSTP1 are involved in the detoxification of a broad range of toxic substances. Genetic polymorphisms in these genes have been studied intensively for their potential role in cancer susceptibility and drug response. In Caucasians, the enzyme activity of GSTM1 and GSTT1 is absent in approximately 50 and 15% of the population, respectively, due to deletions of both chromosomal copies of the genes. A trimodal phenotype pattern exists in which individuals with two, one, or no functional genes are fast, intermediate, or slow "conjugators," respectively. Most studies investigating the effect of the GSTM1 and GSTT1 deletions do not distinguish between fast and intermediate conjugators because the applied genotyping assays only detect if at least one copy of either gene is present. We present a multiplex PCR assay that detects if an individual has none, one, or two copies of the GSTM1 and GSTT1 genes and simultaneously detects the allelic status of the GSTP1 Ile105Val genetic variant. A total of 200 Danes, 100 Somalis, and 100 Greenlanders were genotyped. This multiplex PCR assay enables future large-scale studies to investigate the role of GSTs.