Analysis of clinical phenotypes of compound heterozygotes of Hb J-Bangkok and β-thalassemia / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To analyze hematological characteristics of compound heterozygotes of Hb J-Bangkok and β-thalassemia, and to explore the influence of Hb J-Bangkok on the phenotype of β-thalassemia.</p><p><b>METHODS</b>Peripheral blood samples from a patient carrying Hb J-Bangkok and a β-thalassemia mutation, her family members and three sporadic Hb J-Bangkok carriers were collected. RBC analysis and hemoglobin electrophoresis were performed. Genotypes of α- and β-globin genes were analyzed.</p><p><b>RESULTS</b>The father of the proband and the three sporadic cases were single carriers of Hb J-Bangkok. All of them were asymptomatic and have normal hematological parameters except for an abnormal hemoglobin band detected on hemoglobin electrophoresis. The proband was a compound heterozygote for Hb J-Bangkok and β-thalassemia mutation IVS-Ⅱ-654. She presented typical β-thalassemia trait, featuring hypochromic microcytic anemia and increased Hb A₂ level. An abnormal hemoglobin band was also detected.</p><p><b>CONCLUSION</b>Carriers of Hb J-Bangkok alone are asymptomatic. Co-existence of Hb J-Bangkok and β-thalassemia may not aggravate the phenotype. Therefore, couples with one carrying Hb J-Bangkok and another carrying a β-thalassemia mutation do not require prenatal diagnosis.</p>

Analysis of hematological characteristics on the 79 co-inheritance of α-thalassemia and β-thalassemia carriers in Guangxi / 中华血液学杂志

<p><b>OBJECTIVE</b>To investigate the hematological characteristics of co-inheritance of α-thalassemia (α-thal) and β-thalassemia (β-thal) and to survey the incidence of co-inheritance of α-thal and β-thal in Guangxi.</p><p><b>METHODS</b>DNA samples from 370 primary and middle school students who were β-thal carriers in Guangxi were further processed for the α-goblin gene mutation screening, and were grouped based on the genotype of β- and α-goblin gene. The hematological indexes to the different groups were compared by One-way ANOVA.</p><p><b>RESULTS</b>Of the total 370 β-thal carriers, 79 were found to carry α-thal, which gave a frequency of 21.35% for β-thal carriers and 1.36% for coincidence of these two common disorders in the local population. As expected, the 79 patients presented very variable α-globin alterations in combination with β-globin mutations, showing 31 genotype combined with the coincidence of both Hb disorders. Except the genotypes of 3 β-thal heterozygotes combined with ααα(anti3.7) triplication and 2 β-thal carriers with IVS-II-654(C→T)/N combined-α(3.7)/αα presented the phenotype of thalassemia intermedia, and other 74 carriers with co-inheritance of α-thal and β-thal all presented the phenotype of β-thal trait. There were significant differences between β-thal heterozygotes and the carriers with a co-inheritance of both β+α(0) thal in MCH, MCV and Hb. In addition, there existed significant difference between the carriers with a co-inheritance of both β+α(+) thal and a co-inheritance of both β+α(0) thal in MCV, MCH and Hb.</p><p><b>CONCLUSION</b>Compared to that of β-thal heterozygotes, the carriers with a co-inheritance of α-thal and β-thal had slighter phenotype with hematological characteristics. It's difficult to distinguish the double heterozygotes with the co-inheritance of α-thal and β-thal from β-thal heterozygotes by hematological indexes, the molecular diagnosis should be performed.</p>

Analysis of the phenotype-genotype relationship of Hb Constant Spring / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To analyze the genotype-phenotype correlations in the Hb Constant Spring (HbCS) carriers, and to investigate the effect of HbCS on hematologic parameters.</p><p><b>METHODS</b>Complete blood cell count and hemoglobin electrophoresis analyses were performed in 125 HbCS cases. The α-and β-thalassemia mutations were determined by reverse dot-blotting and Gap-PCR.</p><p><b>RESULTS</b>The presence of the SEA deletion or Hb Quong Sze (HbQS) with HbCS leads to HbH-CS disease. There was significant difference between HbH-CS and αCSα/-α, HbH-CS and αCSα/αα in the hematological parameters. The genotype of αCSα/-α or αα/αCSα had slight effect on hematological parameters. When the Hb Constant Spring mutation co-existed with heterozygous β-thalassemia, the hematological characteristics of β-thalassemia was presented. Only 57.6% of carriers with HbCS were detected by hemoglobin electrophoresis.</p><p><b>CONCLUSION</b>The cases with co-existence of HbCS trait and other α-thalassemia trait, or β-thalassemia trait, showed variation in their red blood cell parameters. For such compound heterozygotes for HbCS and other α- or β-thalassaemia mutations, which were usually misdiagnosed in clinical screening by hemoglobin electrophoresis, accurate diagnose can be made by molecular diagnosis.</p>

Identification of a missense mutation in SEDL gene from a Chinese family with X-linked spondyloepiphyseal dysplasia tarda / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To identify the SEDL gene mutation in a Chinese family with X-linked spondyloepiphyseal dysplasia tarda (SEDL) and to establish a genotyping assay for rapid diagnosis of this X-linked recessive disorder.</p><p><b>METHODS</b>Clinical diagnoses were made based on physical examination, radiological examination and pedigree analysis for this family. Four primer pairs flanking the SEDL exons 3-6 including their exon/intron boundaries were designed. A rapid genotyping assay based on denaturing high performance liquid chromatography (DHPLC) was established to screen the point mutations of the SEDL gene. Genomic DNA was extracted by standard methods from 18 members in the three generations of the pedigree and subjected to PCR-denaturing high performance liquid chromatography (PCR-DHPLC) assay followed by direct DNA sequencing.</p><p><b>RESULTS</b>A c.218C>T mutation in exon 4 of the SEDL gene, which resulted in a substitution of serine 218 with leucine, was identified in this family. Among the 18 members, 3 patients, 5 obligate female carriers and 2 unmarried young females were found to have the missense mutation, and other 8 healthy individuals were not detected to carry the mutation, in which genotype-phenotype correlations were well established in each member investigated in this family.</p><p><b>CONCLUSION</b>A c.218C>T missense mutation in the SEDL gene was firstly reported in Chinese population and the results of this study expand the spectrum of SEDL mutations. The PCR-DHPLC assay is a useful tool to rapidly detect the SEDL mutation in clinical and prenatal diagnosis.</p>

A community-based genetic screening of large-scale population and prenatal diagnosis for alpha and beta thalassemia in Zhuhai city of Guangdong province / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To describe a community-based model for prevention and control of severe alpha and beta thalassemias in Zhuhai city of Guangdong province.</p><p><b>METHODS</b>Couples for premarital medical examination or regular healthcare examination in pregnancy were enrolled in this prospective screening program, which was supported by the two-level network composed of 6 local hospitals for testing thalassemias and follow-up for genetic counseling. A conventional heterozygote screening strategy was used to determine alpha and beta thalassemia traits in women and their partners according to the standard procedures of hematological phenotype analysis. Then confirmative diagnosis of alpha and beta thalassemia was performed on those couples suspected at-risk for severe thalassemia by using the PCR-based molecular diagnostic assays. The couples at-risk for severe thalassemia were counseled and offered prenatal diagnosis and termination of pregnancy in case of an affected fetus.</p><p><b>RESULTS</b>During the period between January 1998 and December 2005, the screened records included 85522 young females and their partners for premarital screening and 10439 pregnant women for prenatal screening, with 71.38% coverage of total population recorded in this city for premarital screening. Six thousands five hundreds and sixty-three individuals in total were found to be the carriers of thalassemias, with 4312 for alpha thalassemia (4.5%) and 2251 for beta thalassemia (2.3%), respectively. One hundred and forty-eight couples were diagnosed to be at-risk for thalassemias, including 103 for alpha thalassemia and 45 for beta thalassemia, respectively. Successful prenatal diagnosis was made for 142 (98 for alpha thalassemia and 44 for beta thalassemia) out of 148 (95.9%) pregnancies at-risk for severe thalassemias. Twenty-three cases of hydrops fetalis, 4 of Hb H diseases and 14 of beta thalassemia were identified. All 41 pregnancies with affected fetuses were voluntarily terminated. Thus, this has led to a marked decrease of severe thalassemia syndrome since the program started.</p><p><b>CONCLUSION</b>We presented the first community-based prospective screening program in China for control of alpha and beta thalassemia in Zhuhai city with a population of 1.29 million through premarital or prenatal screening. This model could be used for control of thalassemias and other hemoglobinopathies in other regions of China and also in other developing countries.</p>

Rapid molecular diagnosis of trisomy 21 using the PCR-STR-SSCP technique / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>Developing a PCR-based method to diagnose trisomy 21 directly by alternative detection of the SSCP profiles of the STR fragments amplified.</p><p><b>METHODS</b>The DNA samples from 19 trisomy 21 patients, 3 at-risk fetuses of trisomy 21 and a total of 44 samples from their parents as controls were drawn for this study, in which the trisomy 21 was determined by G-band karytyping. Two polymorphic STR at D21S11 and D21S1411 served as the gene markers, and two separate PCR-amplified primers were designed.The STR-amplicons denatured were subjected to polyacrylamide gel electrophoresis for SSCP analysis.</p><p><b>RESULTS</b>This assay can identify three STR fragments representing parents' chromosome 21 by detecting the electrophoresis separation profiles of PCR-amplified fragments. With the use of this assay, the authors analyzed the 22 cases of trisomy 21;accurate diagnoses were made except for one case in which the electrophoresis pattern at D21S11 site could not present the diagnostic information because of the homozygous state of this family. The 3 at-risk fetuses were found to be the trisomy 21 patients, followed by confirmation of the results by G-band karytyping of aborted samples.</p><p><b>CONCLUSION</b>The present PCR-STR-SSCP assay can be applied as a simple, rapid and accurate method in the prenatal diagnosis and genetic screening of trisomy 21.</p>

Accurate and rapid prenatal diagnosis of beta-thalassemia by a multiplex primer extension and denaturing high-performance liquid chromatography technique / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To develop a primer-extension in combination with denaturing high-performance liquid chromatography (PE-DHPLC)-based assay for prenatal diagnosis of the five most common beta-thalassemia mutations in Chinese.</p><p><b>METHODS</b>The human beta-globin gene fragment was amplified by PCR, followed by a multiple PE reaction specific for each five mutations. Then the PE product mixtures were separated for genotyping of beta-globin gene mutations using fully-denaturing DHPLC analysis.</p><p><b>RESULTS</b>In a blind study, prenatal diagnosis was performed on thirty-six at-risk families for beta-thalassemia major. Reverse dot blot (RDB) analysis was used to validate each result, showing an accuracy rate of 100% for PE-DHPLC in a total of 108 samples tested. Overall, by PE-DHPLC analysis, the authors could identify the genotypes involving the five mutations and normal alleles corresponding to 94.4% (102/108) and actually make final decision for prenatal diagnosis covering 97.2% (35/36).</p><p><b>CONCLUSION</b>The PE-DHPLC protocol can be a simple, rapid, and highly accurate assay in the prenatal detection of common beta-thalassemia mutations.</p>

Rapid diagnosis of non-deletion alpha-thalassemias by reverse dot blot / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To establish a rapid and convenient method of reverse dot blot (RDB) analysis for detecting the point mutations of non-deletion alpha-thalassemia in Chinese.</p><p><b>METHODS</b>Label biotin to primers and amplify human alpha2 globin gene selectively, then hybridize PCR products with a set of oligonucleotide probes immobilized on strips, and develop colour to detect non-deletion alpha-thalassemia defects.</p><p><b>RESULTS</b>The PCR system using biotin-labeled primers could specifically amplify a 1085 bp fragment of human alpha2 globin gene which encompasses all six alpha-thalassemia mutations. After being hybridized with sequence-specific oligonucleotide probes and colour development, it could simultaneously identify all six types of non-deletion alpha-thalassemias encountered in Chinese.</p><p><b>CONCLUSION</b>This method does not need semi-nested PCR, and the products amplified by biotinylated primers can be used directly to hybridize with the probes on strips under the identical conditions of hybridization. So, it is a specific and multiplex detection assay for screening non-deletion alpha-thalassemia defects in Chinese.</p>

Use of capillary electrophoresis to determine hemoglobin A2 in healthy adults and alpha- and beta-thalassemia carriers / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To evaluate the capillary isoelectric focusing (CIEF) method for the estimation of blood hemoglobin A2 (Hb A2) concentrations in routine thalassemia screening.</p><p><b>METHODS</b>A total of 105 samples from healthy adults and 93 samples with positive phenotypes were collected by routine thalassemia screening. CIEF was compared with Helena spife combo electrophoresis system for Hb A2 measurement and its precision and reproducibility were tested by analyzing intra-assay or inter-assay coefficient of variations(CVs). The reliability and veracity of Hb A2 measurement by CIEF for the detection of alpha- and beta- thalassemia including Hb E were evaluated by genotyping of 93 consecutive samples for routine thalassemia screening.</p><p><b>RESULTS</b>By us e of CIEF for measurement of Hb A2 in a local healthy adult population, the range of reference value(3.59%-5.23%) was obtained. The results of CIEF showed good linearity relation to that of conventional Hb electrophoresis assay. All thalassemia carriers (43 cases of alpha-thals and 44 of beta-thals) or Hb E carriers (6 cases) presumptively identified by the present CIEF for the quantification of Hb A2, combined with routine RBC parameters for indicating microcytosis and hypochromia were confirmed to be the heterozygous or compound heterozygous defects of alpha- or beta- globin gene by molecular diagnosis, without any false positive or false negative results.</p><p><b>CONCLUSION</b>The measurement of Hb A2 by CIEF method is rapid, precise and reproducible; it could be used in routine screening for alpha- or beta- thalassemia.</p>

A rare transcription mutation (-90 C-->T) in a Chinese family with beta-thalassemia / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To identify a rare transcription mutation (C-->T) at position -90 of the beta-globin gene previously unreported in the beta-thalassemia carriers from a Chinese family.</p><p><b>METHODS</b>In phenotype analysis, standard hematological techniques were used to measure RBC counts and Hb concentration. Reverse dot blot (RDB) analysis, which can simultaneously detect 18 known types of beta-thalassemia mutations in Chinese, was used to scan beta-globin gene mutations. DNA sequence analysis of the entire human beta-globin gene was performed to characterize the underlying causative mutation of the sample and to identify its genotype. A semi-quantitative RT-PCR method was used to measure beta-globin gene expression in the form of mRNA from the subjects.</p><p><b>RESULTS</b>The proband, his brother and his mother presented a typical beta-thalassemic trait with reduced mean corpuscular volume (MCV, 68.2-73.6 fL) and elevated level of Hb A(2) (5.7%-6.4%) but no known beta-thalassemia mutations were found in the samples by RDB analysis. DNA sequencing of the beta-gene region of these three samples revealed heterozygosity for the C-->T substitution at position -90 within proximal CACCC box of the beta-globin gene promoter element, which was previously unreported in the Chinese population. Analysis of mRNA from the positive carriers demonstrated that the mutant beta-globin gene significantly reduced beta-globin transcription (mutants: 2.233 +/- 0.01 vs normal: 3.779+/-1.19; 95%CI: 3.060, 4.499), showing a level comparable with that of the other beta-thalassemia heterozygotes (2.110+/-0.53, 95%CI: 1.732, 2.488).</p><p><b>CONCLUSION</b>A rare transcriptional mutation that led to beta-thalassemia in Chinese population has been characterized. The findings enrich knowledge of the mutation spectrum of beta-thalassemia.</p>