A simple approach to prenatal diagnosis of beta-thalassemia in a geographic area where multiple mutations occur

We describe a simple approach for detecting beta-thalassemia mutations in geographic areas such as southern China where multiple mutations are known to occur. Segments of the beta-globin gene were amplified in vitro by using the polymerase chain reaction. Dot blot hybridization of the amplified DNA with oligonucleotide probes corresponding to the six mutations found in southern China could directly identify the mutations causing beta-thalassemia in the affected families. The increased number of target sequences after amplification allows the use of 35S-labeled probes, which are reusable for up to 3 months. The mutations can be determined in two days.     

Factors associated with variability in response of diabetic macular oedema after intravitreal triamcinolone

Purpose:  To identify factors associated with variability in anatomical and functional response of diabetic macular oedema (DMO) after 4 mg of intravitreal triamcinolone acetonide (ivTA), and for recurrence of macular oedema.
Design:  Pooled analysis of individual data from two randomized controlled trials.
Methods:  This was a multicentre study involving 107 eyes with DMO administered 4 mg ivTA. Predictive factors for response to treatment were evaluated with linear regression analysis. Factors associated with time to recurrence of oedema were studied with Cox proportional hazards modelling. Main outcome measures were maximum improvement in optical coherence tomography (OCT)-measured central foveal thickness (CFT) and best-corrected visual acuity (BCVA), final CFT and BCVA at 12 months and time to oedema recurrence.
Results:  Greater reduction of retinal thickening occurred in eyes with worse baseline thickening ( P  < 0.001). There was also greater improvement of visual acuity in eyes with poorer preoperative BCVA levels ( P  < 0.001). Age, duration of oedema and previous macular laser treatment had no significant effect on maximal BCVA or CFT improvement. Eyes given 4 mg triamcinolone alone were more likely to develop recurrence of oedema at 12 months than those given a combination of 4 mg triamcinolone plus sequential laser (hazard ratio 2.60 [95% confidence interval: 1.45–4.67]).
Conclusion:  Baseline OCT-measured retinal thickening and BCVA are important predictors of maximal anatomical and functional response of DMO to ivTA, respectively. Combination treatment strategy using sequential laser therapy may have a role in delaying recurrence of oedema after triamcinolone.     

Apoptosis in the early human placental bed and its discrimination from necrosis using the in-situ DNA ligation technique

Extensive areas of necrosis are present in the early human placental bed. Our aim was to determine whether apoptosis is also a feature. A method was therefore required to differentiate unequivocally necrosis and apoptosis. Initially, terminal deoxynucleotide transferase-mediated dUTP nick-end labelling was used to visualize apoptotic cells. However, non-specific labelling, probably due to free DNA released by necrotic cells, was excessive; thus, in-situ DNA ligation was employed. In this technique, two DNA fragments with single-base 3' overhangs and blunt- ends were labelled with a fluorochrome and then ligated to the DNA breaks on the sections. Immunolabelling for cytokeratin or leukocyte common antigen was performed to determine the phenotype of apoptotic cells identified by the in-situ DNA ligation technique. A proportion of the dying cells was confirmed to be trophoblasts. No co-localization with leukocyte common antigen was found in this region, suggesting that maternal macrophages and natural killer cells (CD56+) were not dying by apoptosis in significant numbers. In conclusion, in-situ DNA ligation in association with immunocytochemistry can readily distinguish apoptosis from necrosis in the placental bed. The results suggest that a proportion of invading trophoblast cells are eliminated by apoptosis in early pregnancy.      

Isolation of hybrid cell clones that contain deletion and non-deletion defects of alpha-thalassemia in man

We have succeeded in isolating hybrid mouse erythroleukemia cell clones from a patient with hemoglobin H disease, which exhibit either deletion or nondeletion mutations of the human alpha-globin genes. Analysis of one of these hybrid clones that had retained a human chromosome 16 from the patient's cells showed that both human alpha-globin had been deleted. Several clones of another hybrid cell had retained a human chromsome 16 from the patient's cells, which contained both human alpha- globin genes on an EcoRI fragment of 23 kilobases (kb). These latter hybrid clones showed the presence of human alpha-globin chains at detectable but low levels. These studies show that there are two different types of human chromosome 16 in this patient and that the nondeletion mutation of human alpha-globin genes leading to hemoglobin H diseases in this patient acts in cis to the two alpha-globin genes remaining in his cells. The close correlation between the pattern of human alpha-globin gene expression in the patient and in the hybrid cells suggests that this method of transfer of human globin genes to rodent cells will be a useful one for study of mutations affecting the expression of differentiated genes that lead to disease in man.     

The frequency of an IL-18-associated haplotype in Africans

Variation within the gene for the proinflammatory cytokine interleukin (IL)-18 has been associated with inter-individual differences in levels of free protein and disease risk. We investigated the frequency of function-associated IL18 gene haplotypes in an extensive sample (n=2357) of African populations from across the continent. A previously identified five tagging SNP (single-nucleotide polymorphism) haplotype (here designated hGTATA), known to be associated with lower levels of IL-18, was observed at a frequency of 27% in a British population of recent European ancestry, but was found at low frequency (<8%) or completely absent in African populations. Potentially protective variants may, as a consequence, be found at low frequency in African individuals and may confer a difference in disease risk.     

脊髓型颈椎病患者颈椎间盘白细胞介素6和肿瘤坏死因子α水平与日本骨科学会颈髓功能评分的相关性

目的:检测脊髓型颈椎病患者颈椎间盘组织中白细胞介素6、肿瘤坏死因子α的水平,分析其与日本骨科协会颈髓功能评分(JOA)的相关性,探讨免疫反应在脊髓型颈椎病发病机制中的作用。方法:病例来源于2005-11-10/2006-06-30在安徽医科大学第一附属医院骨科住院患者,27例脊髓型颈椎病患者为脊髓型颈椎病组,另外16例无颈椎病史的颈椎外伤患者为对照组。两组均经颈椎前路手术中切取的颈椎间盘组织,应用放射免疫分析法测定两组椎间盘组织中白细胞介素6和肿瘤坏死因子α的水平。脊髓型颈椎病患者组依据JOA评分分成重、中度组(0～12分,20例)和轻度组(13～17分,7例)。结果:43例全部进入结果分析。①脊髓型颈椎病组颈椎间盘组织内白细胞介素6、肿瘤坏死因子α的水平均明显高于对照组[白细胞介素6:(34.521±18.592),(15.041±6.562)ng/L;肿瘤坏死因子α:(6.071±1.912),(3.143±0.630)pmol/L;P均<0.01]。②脊髓型颈椎病患者重、中度组椎间盘中肿瘤坏死因子α水平明显高于轻度组(P<0.01)。结论:①脊髓型颈椎病患者的颈椎间盘组织内白细胞介素6、肿瘤坏死因子α的分泌增多。②脊髓型颈椎病患者颈椎间盘中肿瘤坏死因子α的水平与脊髓功能有相关性,提示脊髓型颈椎病患者临床症状的轻重与免疫反应之间存在着一定的关系。