Next-generation sequencing improves molecular epidemiological characterization of thalassemia in Chenzhou Region, P.R. China.

OBJECTIVES: Thalassemia is a highly prevalent monogenic inherited disease in southern China. It is important to collect epidemiological data comprehensively for proper prevention and treatment. METHODS: In this study, blood samples collected from 15 807 residents of Chenzhou were primarily screened by hematological tests. A total of 3973 samples of suspected thalassemia carriers were further characterized by combined next-generation sequencing (NGS) and Gap-PCR. RESULTS: In total, 1704 subjects were diagnosed as thalassemia carriers with a total prevalence rate of 10.78%, including 943 α-thalassemia carriers, 708 ß-thalassemia carriers, and 53 composite α and ß-thalassemia carriers. The prevalence rates of α-thalassemia, ß-thalassemia, and composite α and ß-thalassemia were 5.97%, 4.48%, and 0.34%, respectively. Meanwhile, we characterized 19 α-thalassemia variations and 21 ß-thalassemia variations in thalassemia carriers. Approximately 2.88% of thalassemia carriers would be missed by traditional genetic analysis. In addition, four novel thalassemia mutations and one novel abnormal hemoglobin mutation were identified. CONCLUSIONS: Our data suggest a high prevalence of thalassemia and a diverse spectrum of thalassemia-associated variations in Chenzhou. Also, combined NGS and Gap-PCR is an effective thalassemia screening method. Our findings might be helpful for prevention and treatment of thalassemia in this region.

Primer-introduced restriction analysis polymerase chain reaction method for non-invasive prenatal testing of ß-thalassemia.

We have developed a new method for non-invasive prenatal testing (NIPT) of paternally inherited fetal mutants for ß-thalassemia (ß-thal). Specially designed primer-introduced restriction analysis-polymerase chain reaction (PIRA-PCR) were used to detect four major mutations [IVS-II-654, HBB: c.316-197C > T; codon 17 (A > T), HBB: c.52A > T; -28 (A > G), HBB: c.-78A > G and codons 41/42 (-TTCT), HBB: c.126_129delCTTT] causing ß-thal in China. The PIRA-PCR assay was first tested in a series of mixed DNA with different concentrations and mixed proportions. Subsequently, this assay was further tested in 10 plasma DNA samples collected from pregnant women. In the DNA mixture simulation test, the PIRA-PCR assay was able to detect 3.0% target genomic DNA (gDNA) mixed in 97.0% wild-type gDNA isolated from whole blood. For plasma DNA testing, the results detected by PIRA-PCR assay achieved 100.0% consistency with those obtained from the amniocentesis analysis. This new method could potentially be used for NIPT of paternally inherited fetal mutants for ß-thal.

A short-read multiplex sequencing method for reliable, cost-effective and high-throughput genotyping in large-scale studies.

Accurate genotyping is important for genetic testing. Sanger sequencing-based typing is the gold standard for genotyping, but it has been underused, due to its high cost and low throughput. In contrast, short-read sequencing provides inexpensive and high-throughput sequencing, holding great promise for reaching the goal of cost-effective and high-throughput genotyping. However, the short-read length and the paucity of appropriate genotyping methods, pose a major challenge. Here, we present RCHSBT-reliable, cost-effective and high-throughput sequence based typing pipeline-which takes short sequence reads as input, but uses a unique variant calling, haploid sequence assembling algorithm, can accurately genotype with greater effective length per amplicon than even Sanger sequencing reads. The RCHSBT method was tested for the human MHC loci HLA-A, HLA-B, HLA-C, HLA-DQB1, and HLA-DRB1, upon 96 samples using Illumina PE 150 reads. Amplicons as long as 950 bp were readily genotyped, achieving 100% typing concordance between RCHSBT-called genotypes and genotypes previously called by Sanger sequence. Genotyping throughput was increased over 10 times, and cost was reduced over five times, for RCHSBT as compared with Sanger sequence genotyping. We thus demonstrate RCHSBT to be a genotyping method comparable to Sanger sequencing-based typing in quality, while being more cost-effective, and higher throughput.

Characterization of 27 novel HLA class II alleles from China marrow donor program.

Twenty-seven novel human leukocyte antigen (HLA) class II alleles in Chinese Marrow Donors are described: 10 HLA-DRB1 alleles and 17 HLA-DQB1 alleles.

Identification of a novel HLA-C allele, HLA-C*04:82, by sequence-based typing in a Chinese individual.

A novel HLA allele, C*04:82, was identified in a Chinese individual, which has nine nucleotides insertion compared with the closest matching allele HLA-C*04:01:01 in exon 5, resulting in three amino acid insertions: 301 Ala, 302 Val and 303 Leu.