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.

A novel 223 kb deletion in the beta‐globin gene cluster was identified in a Chinese thalassemia major patient

“…The 5′ breakpoint of these two deletions was very similar to this 223 kb deletion, both with a similar 5′ breakpoint to this case (Figure A). Three mechanisms may contribute to this different HbF level: (a) the 3′ breakpoints of the above two deletions were different from the present case; (b) the expression of HbF is regulated by many genetic modifiers such as XmnI polymorphism, KLF1, BCL11A, HBS1L, and MYB . The NGS data of our cases showed that they were all negative for mutations in these factors, while this information was absent for the carriers of the above two deletions; and (c) the 5′ breakpoint of the 223 kb deletion was not completely consistent in the above two deletions.…”

“…Second, individuals who are compound heterozygous for a large deletion and point mutation would be misdiagnosed as homozygous for the point mutation, such as the proband III 1 in this family. Similar examples were also reported by Shang 2017 . Therefore, molecular screening methods should be prioritized in areas with high incidence of thalassemia to increase the detection rate of genetic mutation carriers.…”

“…Next‐generation sequencing was performed in the proband and was compared with two unrelated normal controls. The entire protein‐coding regions, key regulatory regions, known pathogenic copy number variants (CNVs) and single nucleotide variants (SNVs)/insertion and deletion variants (indels) in the noncoding regions of the beta‐globin gene clusters, and four modifier genes ( KLF1 , BCL11A , HBS1L, and MYB ) were targeted …”

“…Different location and length of deletion may result in different phenotypes. Studies of molecular structure of these large deletions especially the precise breakpoints were difficult, until the invention of next‐generation sequencing (NGS) technology which could relatively accurately estimate the deletion range . Here, we identified a novel large deletion in the beta‐globin gene cluster in a Chinese family and characterized it precisely by NGS.…”

Thalassaemia intermedia caused by coinheritance of a β‐thalassaemia mutation and a de novo duplication of α‐globin genes in the paternal allele

“…Copy number variations (CNVs) of the α‐ and β‐globin gene clusters were analysed by multiplex ligation dependent probe amplification (MLPA) (Hu et al , ). Next generation sequencing (NGS) was also used to confirm the HBA rearrangement region and other mutations of modifier genes (Shang et al , ). To verify the duplication breakpoints, three pairs of primers were designed upstream and downstream of this rearrangement region, based on the NGS results, as follows (Fig C):…”