Non‑invasive prenatal diagnosis of thalassemia through multiplex PCR, target capture and next‑generation sequencing

Abstract

Prenatal clinical detection of thalassemia involves gap-Pcr and reverse dot blot (rdB) analysis of fetal dna acquired through invasive methods. The present study aimed to develop a non-invasive prenatal diagnostic method for thalassemia based on next-generation sequencing (nGS). a total of eight families with proband children with thalassemia were recruited for the study during a subsequent pregnancy. The sequence of the thalassemia genes of the parents and proband were determined using nGS, based on a thalassemia ampliSeq panel. cell-free plasma dna from pregnant women related to the aforementioned proband was analyzed using an nGS panel, based on thalassemia-associated capture probes. Heterozygous single nucleotide polymorphisms within the 10 kb regions flanking exons of the targeted thalassemia genes were acquired using probes or ampliSeq and employed for parental haplotype construction using Trio-based panel sequencing. The fetal haplotype was deduced from the parental haplotypes and relative haplotype dosage, and subsequently validated using gap-Pcr and rdB, based on invasively sampled amniotic fluid. A non-invasive prenatal diagnosis procedure from maternal plasma fetal dna was successfully developed based on haplotype analysis. The deduced haplotypes of eight fetuses were identical to the results of invasive prenatal diagnosis procedures, with an accuracy rate of 100%. Taken together, the present study demonstrated the potential for non-invasive prenatal diagnosis of α-and β-thalassemia using nGS and haplotype-assisted analysis.

Cell-Free Fetal DNA and Non-Invasive Prenatal Diagnosis of Chromosomopathies and Pediatric Monogenic Diseases: A Critical Appraisal and Medicolegal Remarks

“…We can see that most of them were published in 2021 (13 out of 51 studies), likely due to the relatively young age of the investigation technique. Regarding the reviewed studies, 13 are described in the first section concerning the role of cffDNA in the non-invasive prenatal diagnosis of aneuploidies [ 4 , 5 , 6 , 7 , 8 , 9 , 10 , 11 , 12 , 13 , 14 , 15 , 16 ], 7 are described in the second section (CNV diseases) and the remaining 31 are in the third section concerning diseases with monogenic transmission [ 2 , 17 , 18 , 19 , 20 , 21 , 22 , 23 , 24 , 25 , 26 , 27 , 28 , 29 , 30 , 31 , 32 , 33 , 34 , 35 , 36 , 37 , 38 , 39 , 40 , 41 , 42 , 43 , 44 , 45 , 46 ].…”

“…Among hematologic disorders, various papers focused on NIPD of alpha or beta thalassemia [ 19 , 24 , 31 , 41 ]. In the study of D’Souza [ 41 ], the non-invasive approach gave comparable results to those obtained by the conventional invasive fetal sampling methods in 24 cases, with an accuracy of 80.0%.…”

Noninvasive prenatal prediction of fetal haplotype with Spearman rank correlation analysis model

“…Recently, long‐read sequencing methods have been shown used as a more accurate and less complex parental haplotype analysis. HMM (Wang et al, 2017 ) and SPRT (Yang et al, 2020 ) are suitable for families with probands, and methods based on databases and populations are suitable for families without probands.…”

“…Combined with the genotype of the proband, haplotypes of each parent that were transmitted to their firstborn were distinguished from the others not inherited. Haplotypes that were inherited and not inherited by the proband were called H0 and H1, accordingly (Yang et al, 2020 ).…”

Research Progress in Isolation and Enrichment of Fetal Cells from Maternal Blood

“…It can amplify the specific sequence of the entire fetal genome, obtain enough DNA for analysis, and use a minimum number of cells and quantitative analysis to identify the source of the cells. Yang et al [34] used multiplex PCR, target capture, and next-generation sequencing to perform noninvasive prenatal diagnosis of thalassemia. Guissart et al [35] used a universal detection method for indirect diagnosis of cystic fibrosis (CF) based on fluorescence multiplex PCR and large and small fragment analysis.…”