Noninvasive Prenatal Test for β-Thalassemia and Sickle Cell Disease Using Probe Capture Enrichment and Next-Generation Sequencing of DNA in Maternal Plasma

Abstract

Background Noninvasive prenatal testing (NIPT) of chromosomal aneuploidies based on next-generation sequencing (NGS) analysis of fetal DNA in maternal plasma is well established, but testing for autosomal recessive disorders remains challenging. NGS libraries prepared by probe capture facilitate the analysis of the short DNA fragments plasma. This system has been applied to the β-hemoglobinopathies to reduce the risk to the fetus. Method Our probe panel captures >4 kb of the HBB region and 435 single-nucleotide polymorphisms (SNPs) used to estimate fetal fraction. Contrived mixtures of DNA samples, plasma, and whole blood samples from 7 pregnant women with β-thalassemia or sickle cell anemia mutations and samples from the father, sibling, and baby or chorionic villus were analyzed. The fetal genotypes, including point mutations and deletions, were inferred by comparing the observed and expected plasma sequence read ratios, based on fetal fraction, at the mutation site and linked SNPs. Accuracy was increased by removing PCR duplicates and by in silico size selection of plasma sequence reads. A probability was assigned to each of the potential fetal genotypes using a statistical model for the experimental variation, and thresholds were established for assigning clinical status. Results Using in silico size selection of plasma sequence files, the predicted clinical fetal genotype assignments were correct in 9 of 10 plasma libraries with maternal point mutations, with 1 inconclusive result. For 2 additional plasmas with deletions, the most probable fetal genotype was correct. The β-globin haplotype determined from linked SNPs, when available, was used to infer the fetal genotype at the mutation site. Conclusion This probe capture NGS assay demonstrates the potential of NIPT for β-hemoglobinopathies.

Insights in Thalassemia: From Genomics to Clinical Practice

“…Notably, the gene-corrected β-thalassemia iPSCs restored HBB expression and reduced reactive oxygen species production compared to un-corrected group, implying that CRISPR/Cas9 system had greatly improve the hematopoietic differentiation efficiency of β-thalassemia iPSCs (45).…”

“…NGS provides efficient means of constructing parental haplotypes to assess fetal genotypes, i.e., by removing the needs of complicated haplotypic block/proband analysis (44). Although errors from maternal background contaminations still remains an issue, the combined NGS-RHDO method has been demonstrated to provide accurate fetal risk assessments concurrent with the results obtained through invasive prenatal diagnosis procedures (45)(46)(47). Further, a recently developed NGS-based NIPD by Yang et al called the cffDNA barcode enabled single molecule test (cfBEST) is a counting system to quantitatively deduce maternal and fetal genotypes (48).…”

Applications of next generation sequencing in the screening and diagnosis of thalassemia: A mini-review

“…NGS provides efficient means of constructing parental haplotypes to assess fetal genotypes, i.e., by removing the needs of complicated haplotypic block/proband analysis (44). Although errors from maternal background contaminations still remains an issue, the combined NGS-RHDO method has been demonstrated to provide accurate fetal risk assessments concurrent with the results obtained through invasive prenatal diagnosis procedures (45)(46)(47). Further, a recently developed NGS-based NIPD by Yang et al called the cffDNA barcode enabled single molecule test (cfBEST) is a counting system to quantitatively deduce maternal and fetal genotypes (48).…”