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Objective: To perform molecular diagnosis and pedigree analysis for one case with α-thalassemia who does not conform to the genetic laws, and explore the effects of a newly discovered rare mutation ( HBA2:c.*12G>A ) on clinical phenotypes., Methods: Blood samples of the proband and her family members were collected for blood routine analysis, and the hemoglobin components were analyzed by capillary electrophoresis. The common α- and β-globin gene loci in Chinese population were detected by conventional techniques (Gap-PCR, RDB-PCR). The α-globin gene sequences ( HBA1, HBA2 ) were analyzed by Sanger sequencing., Results: By analyzing the test results of proband and her family members, the genotype of the proband was -α 3.7 / HBA2:c.*12G>A , her father was HBA2:c.*12G>A heterozygous mutation carrier., Conclusion: This study identifies a rare α-globin gene mutation ( HBA2:c.*12G>A ) that has not been reported before. It is found that heterozygous mutation carriers present with static α-thalassemia.

Objective: To investigate two cases of rare pathogenic genes, initiation codon mutations in HBA2 gene, combined with Southeast Asian deletion and their family members to understand the relationship of HBA2:c.2T>C and HBA2:c.2delT mutations with clinical phenotype., Methods: The peripheral blood of family members was obtained for blood cell analysis and capillary electrophoresis hemoglobin analysis. Gap-PCR and reverse dot blotting (RDB) were used to detect common types of mutations in ɑ-thalassaemia gene. Sanger sequencing was used to analyze HBA1 and HBA2 gene sequence., Results: Two proband genotypes were identified as --SEA/αα with HBA2:c.2T>C and --SEA/αα with HBA2:c.2delT . HBA2:c.2T>C/WT and HBA2:c.2delT/WT was detected in family members. They all presented with microcytic hypochromic anemia., Conclusion: When HBA2:c.2T>C and HBA2:c.2delT are heterozygous that can lead to static α-thalassemia phenotype, and when combined with mild α-thalassemia, they can lead to the clinical manifestations of hemoglobin H disease. This study provides a basis for genetic counseling.

Objective: To proceed the clinical evaluation of DNA microarray for thalassemia gene detection., Methods: Peripheral blood samples of 166 thalassemia gene test subjects were collected and tested for thalassemia genes by microarray chip method and Gap-PCR method combined with PCR-reverse dot blot hybridization method according to double-blind control test. The specificity, sensitivity, positive predictive value, negative predictive value, and total coincidence rate of the microarray chip method were evaluated. When the two methods were inconsistent, multiplex ligation dependent probe amplification (MLPA) was used to verify the deletional α-thalassemia., Results: Compared with Gap-PCR method, specificity, sensitivity, positive predictive value, negative predictive value, Youden index, and total coincidence rate of microarray chip method was 100% (70/70), 96.88% (93/96), 100% (93/93), 95.89% (70/73), 0.969, and 97.59% (162/166), respectively, while compared with PCR-reverse dot blot hybridization method was 100% (125/125), 100% (41/41), 100% (41/41), 100% (125/125), 1, and 100% (166/166), respectively., Conclusion: The microarray chip method for α-thalassemia gene detection shows the advantages of high specificity, sensitivity, and throughput.

Objective: To perform dried blood spots thalassemia gene detection in patients with positive blood phenotypes by microarray technology, and evaluate its value in clinical detection., Methods: DNA samples were extracted from dried blood spots of 410 patients. Microarray technology was used to detect 3 deletion and 3 non-deletion types of α-thalassemia and 19 β-thalassemia point mutations which were common gene mutions in China., Results: There were 357 positive cases in all the 410 tested samples with the positive rate 87.07%, among which 299 cases (72.93%) carried deletion or point mutations of α-thalassemia, 29 cases (7.07%) carried point mutations of β-thalassemia and 29 cases (7.07%) carried gene mutations of complex αβ-thalassemia syndrome. The mutations of α-thalassemia were involved with -- SEA heterozygous deletion (177 cases, 59.2%), α CS heterozygote (60 cases, 20.07%) and several other genotypes. The common mutations of β- thalassemia were involved with β CD41-42 heterozygote (10 cases, 34.48%) and β CD17 heterozygote (9 cases, 31.03%). The mutations of complex αβ-thalassemia syndrome were mainly involved with -- SEA /αα+β CD17 /β N (7 cases, 24.14%), α CS α/αα + β CD41-42 /β N (3 cases, 10.34%) and -α 4.2 /αα + β CD17 /β N (3 cases, 10.34%)., Conclusion: The most common genetic mutations are -- SEA for α-thalassemia and CD41-42 for β-thalassemia in Liuzhou, Guangxi Zhuang Autonomous Region. A and β-thalassemia can be detected at the same time by microarray chip technology in a high throughput manner.

The aim of this study was to investigate a family with nonhomologous sequence recombination of HBA1 and HBA2 genes and provide a favorable basis for genetic counseling and eugenics. Peripheral blood of family members was collected. Hematological parameters were determined by an automated cell counter and hemoglobin (Hb) analysis was performed using high performance liquid chromatography (HPLC). Villus samples were taken for prenatal diagnosis (PND). Gap-polymerase chain reaction (gap-PCR) and reverse dot-blot were used for thalassemia genotyping. DNA sequencing was used to analyze the gene sequence of HBA1 (α1-globin) and HBA2 (α2-globin). The nonhomologous sequence recombination allelic variant of HBA1 and HBA2 genes were identified, namely, a gene conversion on the HBA2 gene called α12 ( HBA12 ). The α12 allele consists primarily of the HBA2 gene sequence except for a segment of the IVS-II in which HBA2 -specific sequences have been replaced by HBA1 -specific sequences. The following genotypes were observed: - - SEA /αα12 (Southeast Asian deletion), αα/αα12 and α QS α/αα12 (Hb Quong Sze or Hb QS; HBA2 : c.377T>C), and all manifested as small cell hypochromic anemia. To find the α12 allele in the Chinese population and clarify the influence of the α12 allele and its common inheritance with abnormal Hb and α-thalassemia (α-thal) on α-globin gene expression can help guide clinical diagnosis and genetic counseling.

Background: The Southeast Asian deletion (--(SEA)) is the most commonly observed mutation among diverse α-thalassemia alleles in Southeast Asia and South China. It is generally argued that mutation --(SEA), like other variants causing hemoglobin disorders, is associated with protection against malaria that is endemic in these regions. However, little evidence has been provided to support this claim., Results: We first examined the genetic imprint of recent positive selection on the --(SEA) allele and flanking sequences in the human α-globin cluster, covering a genomic region spanning ~410 kb, by genotyping 28 SNPs in a Chinese population consisting of 76 --(SEA) heterozygotes and 138 normal individuals. The pattern of linkage disequilibrium (LD) and the long-range haplotype test revealed a signature of positive selection. The network of inferred haplotypes suggested a single origin of the --(SEA) allele., Conclusions: Thus, our data support the hypothesis that the --(SEA) allele has been subjected to recent balancing selection, triggered by malaria.

Reliable detection of large deletions from cell-free fetal DNA (cffDNA) in maternal plasma is challenging, especially when both parents have the same deletion owing to a lack of specific markers for fetal genotyping. In order to evaluate the efficacy of a non-invasive prenatal diagnosis (NIPD) test to exclude α-thalassemia major that uses SNPs linked to the normal paternal α-globin allele, we established a novel protocol to reliably detect paternal SNPs within the (--(SEA)) breakpoints and performed evaluation of the diagnostic potential of the protocol in a total of 67 pregnancies, in whom plasma samples were collected prior to invasive obstetrics procedures in southern China. A group of nine SNPs identified within the deletion breakpoints were scanned to select the informative SNPs in each of the 67 couples DNA by multiplex PCR based mini-sequencing technique. The paternally inherited SNP allele from cffDNA was detected by allele specific real-time PCR. A protocol for reliable detection of paternal SNPs within the (--(SEA)) breakpoints was established and evaluation of the diagnostic potential of the protocol was performed in a total of 67 pregnancies. In 97% of the couples one or more different SNPs within the deletion breakpoint occurred between paternal and maternal alleles. Homozygosity for the (--(SEA)) deletion was accurately excluded in 33 out of 67 (49.3%, 95% CI, 25.4-78.6%) pregnancies through the implementation of the protocol. Protocol was completely concordant with the traditional reference methods, except for two cases that exhibited uncertain results due to sample hemolysis. This method could be used as a routine NIPD test to exclude gross fetal deletions in α-thalassemia major, and could further be employed to test for other diseases due to gene deletion.