Rapid Targeted Next-Generation Sequencing Platform for Molecular Screening and Clinical Genotyping in Subjects with Hemoglobinopathies

Abstract

Hemoglobinopathies are among the most common autosomal-recessive disorders worldwide. A comprehensive next-generation sequencing (NGS) test would greatly facilitate screening and diagnosis of these disorders. An NGS panel targeting the coding regions of hemoglobin genes and four modifier genes was designed. We validated the assay by using 2522 subjects affected with hemoglobinopathies and applied it to carrier testing in a cohort of 10,111 couples who were also screened through traditional methods. In the clinical genotyping analysis of 1182 β-thalassemia subjects, we identified a group of additional variants that can be used for accurate diagnosis. In the molecular screening analysis of the 10,111 couples, we detected 4180 individuals in total who carried 4840 mutant alleles, and identified 186 couples at risk of having affected offspring. 12.1% of the pathogenic or likely pathogenic variants identified by our NGS assay, which were undetectable by traditional methods. Compared with the traditional methods, our assay identified an additional at-risk 35 couples. We describe a comprehensive NGS-based test that offers advantages over the traditional screening/molecular testing methods. To our knowledge, this is among the first large-scale population study to systematically evaluate the application of an NGS technique in carrier screening and molecular diagnosis of hemoglobinopathies.

Keywords: Clinical genotyping; Hemoglobinopathy; Molecular screening; Next-generation sequencing.

Fig. 1

The sample composition of Group I. A total of 2522 clinical samples with hemoglobinopathies were used in our study. The composition and application of these samples in this study are shown. Among them are included 1182 samples with TM or TI, which were included to evaluate SNP effects on clinical severity. The data from 435 TI/TM samples were collected from a cohort from our previous report, which had been thoroughly sequenced by NGS (Liu et al., 2014). Among the 747 samples, 510 β⁰/β⁰ patients were first used to identify the SNPs that might elevate HbF levels (see Fig. S6).

Fig. 2

The regional and ethnicity distribution of the 20,222 population samples from five provinces in southern China. Among the 20,222 individuals, 4622 were from Guangdong Province, 4834 were from Guangxi Province, 4082 were from Guizhou Province, 2720 were from Hainan Province and 3964 were from Yunnan Province. The ethnic groups of these samples are also listed.

Fig. 3

Comparison of couples identified as being at risk for hemoglobinopathies by using the NGS method and the traditional routine method. A total of 4180 out of 20,222 individuals tested positive with the NGS method. Of these 4180 individuals, 2506 were also identified as positive by the traditional routine method. A total of 1538 individuals were not detected by the routine method because they showed negative phenotypes, and 136 individuals were not detected because their mutations were misdiagnosed or could not be detected by the routine method. Overall, an additional 35 couples were identified as being at risk for α- and β-thalassemia using our NGS assay.