Accurate and up-to-date data on the frequency of haemoglobinopathies among the populations of Guangxi Zhuang Autonomous Region, where haemoglobinopathies are most endemic in China, are required. In our study, a total of 5789 samples obtained from members of the Han, Zhang, and Yao ethnic groups in six geographical areas of Guangxi Province were analysed systematically in terms of both haematological and molecular parameters. The results presented that the total heterozygote frequency of thalassaemias and other haemoglobinopathies was 24.51%, of which 17.55% was due to alpha-thalassaemia, 6.43% to beta-thalassaemia, 0.38% to structural haemoglobin variants, and 0.16% to delta-thalassaemia. The mutational spectrum among the local population for each type of disorder was described, including the first report on the true prevalence of three silent alpha thalassemia defects, -alpha(3.7)/(4.78%), -alpha(4.2)/(1.61%) and Hb Westmead (alpha(WS)alpha/) (1.57%) and of delta-thalassemia resulting from five novel and two rare mutations never before identified in Chinese individuals. Comparison of the frequencies of alpha-globin mutations among the ethnic groups showed that there was a statistically significant difference between the Han (15.71%) and Zhuang (20.12%), and between the Han (15.71%) and Yao (20.84%) ethnic groups. In addition, we have performed the first extensive study of haematological parameters of the Hb Westmead mutation using a group of Chinese subjects with compound heterozygosity for this variant and an alpha-thalassaemia deletion. The knowledge gained in this study will enable us to estimate the health burden in this high-risk population and to elucidate the various genetic alterations that underlie haemoglobinopathies.

Until recently our approach to analyzing human genetic diseases has been to accurately phenotype patients and sequence the genes known to be associated with those phenotypes; for example, in thalassemia, the globin loci are analyzed. Sequencing has become increasingly accessible, and thus a larger panel of genes can be analyzed and whole exome and/or whole genome sequencing can be used when no variants are found in the candidate genes. By using such approaches in patients with unexplained anemias, we have discovered that a broad range of hitherto unrelated human red cell disorders are caused by variants in KLF1, a master regulator of erythropoiesis, which were previously considered to be extremely rare causes of human genetic disease.

The analysis of circulating nucleic acids has revealed applications in the noninvasive diagnosis, monitoring, and prognostication of many clinical conditions. Circulating fetal-specific sequences have been detected and constitute a fraction of the total DNA in maternal plasma. The diagnostic reliability of circulating DNA analysis depends on the fractional concentration of the targeted sequence, the analytical sensitivity, and the specificity. The robust discrimination of single-nucleotide differences between circulating DNA species is technically challenging and demands the adoption of highly sensitive and specific analytical systems. We have developed a method based on single-allele base extension reaction and MS, which allows for the reliable detection of fetal-specific alleles, including point mutations and single-nucleotide polymorphisms, in maternal plasma. The approach was applied to exclude the fetal inheritance of the four most common Southeast Asian ␤-thalassemia mutations in at-risk pregnancies between weeks 7 and 21 of gestation. Fetal genotypes were correctly predicted in all cases studied. Fetal haplotype analysis based on a single-nucleotide polymorphism linked to the ␤-globin locus, HBB, in maternal plasma also was achieved. Consequently, noninvasive prenatal diagnosis in a mother and father carrying identical ␤-thalassemia mutations was accomplished. These advances will help in catalyzing the clinical applications of fetal nucleic acids in maternal plasma. This analytical approach also will have implications for many other applications of circulating nucleic acids in areas such as oncology and transplantation. R ecently, much interest has been focused on the biology and diagnostic applications of nucleic acids that are present in the plasma and serum of humans (1, 2). In particular, fetal DNA has been found to exist in maternal plasma (3). This discovery has facilitated the development of noninvasive prenatal diagnostic approaches based simply on the analysis of a maternal blood sample (4). The noninvasive nature of maternal plasmabased approaches represents a major advantage over conventional methods of prenatal diagnosis, such as amniocentesis and chorionic villus sampling, which are associated with a small but finite risk of fetal loss. However, a technical challenge experienced by many workers in the field relates to the ability to discriminate fetal DNA from the coexisting background of maternal DNA in maternal plasma. During pregnancy, fetal DNA amounts to Ϸ3-6% of the total DNA in maternal plasma (5). Hence, the diagnostic reliability of fetal DNA analysis in maternal plasma depends on the sensitivity and specificity of the analytical system for the detection of fetal-specific markers.Fetal SRY and RHD DNA detection from maternal plasma has reached close to 100% accuracy, as confirmed by many largescale evaluations (6-9). The high level of diagnostic accuracy is attained by the analytical sensitivity contributed by the use of real-time quantitative PCR (5, 10) and the analytical specifici...