Simultaneous detection of target CNVs and SNVs of thalassemia by multiplex PCR and next‑generation sequencing

Fan, Daiming; Yang, Xu; Huang, Lv‐Yin; Ouyang, Guojun; Yang, Xiaobo; Li, Ming

doi:10.3892/mmr.2019.9896

Cited by 7 publications

(5 citation statements)

References 44 publications

(52 reference statements)

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“…The majority of the CNVs are extremely rare and unique to the families in which they have been described. As more of such CNVs are identified with universal screening, there is a need for a comprehensive single methodology akin to next-generation sequencing that can fully characterise breakpoints and genomic recombination events in a single analytical process [32]. Many studies suggest that WGS can be used to accurately locate the breakpoints of large segment deletions/duplications [33].…”

Section: Discussionmentioning

confidence: 99%

A Novel Tool for the Analysis and Detection of Copy Number Variants Associated with Haemoglobinopathies

Minaidou

Tamana

Stephanou

et al. 2022

IJMS

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Several types of haemoglobinopathies are caused by copy number variants (CNVs). While diagnosis is often based on haematological and biochemical parameters, a definitive diagnosis requires molecular DNA analysis. In some cases, the molecular characterisation of large deletions/duplications is challenging and inconclusive and often requires the use of specific diagnostic procedures, such as multiplex ligation-dependent probe amplification (MLPA). Herein, we collected and comprehensively analysed all known CNVs associated with haemoglobinopathies. The dataset of 291 CNVs was retrieved from the IthaGenes database and was further manually annotated to specify genomic locations, breakpoints and MLPA probes relevant for each CNV. We developed IthaCNVs, a publicly available and easy-to-use online tool that can facilitate the diagnosis of rare and diagnostically challenging haemoglobinopathy cases attributed to CNVs. Importantly, it facilitates the filtering of available entries based on the type of breakpoint information, on specific chromosomal and locus positions, on MLPA probes, and on affected gene(s). IthaCNVs brings together manually curated information about CNV genomic locations, functional effects, and information that can facilitate CNV characterisation through MLPA. It can help laboratory staff and clinicians confirm suspected diagnosis of CNVs based on molecular DNA screening and analysis.

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Section: Discussionmentioning

confidence: 99%

A Novel Tool for the Analysis and Detection of Copy Number Variants Associated with Haemoglobinopathies

Minaidou

Tamana

Stephanou

et al. 2022

IJMS

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“…Recently, Yang et al ( 43 ) reported a robust and versatile NGS-based cffDNA allelic molecule counting system termed the cffDNA barcode enabled single molecule test, which was developed for the non-invasive prenatal diagnosis of β-thalassemia. However, these methods mainly aim to identify SNV-associated thalassemia and have not been applied to CNV-associated thalassemia ( 44 ).…”

Section: Discussionmentioning

confidence: 99%

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

Yang

Fan

et al. 2020

Mol Med Rep

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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.

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“…RT was subsequently performed on RNA samples (2 µg) using the PrimeScript™ RT reagent (Takara Bio, Inc., Otsu, Japan), following the manufacturer's protocol. cDNA (1 µl) mixed with Ssofast™ EvaGreen ® Supermix (Bio-Rad Laboratories, Inc., Hercules, CA, USA), DEPC-treated water, forward primer and reverse primer (Sangon Biotech Co., Ltd.) to obtain a 20-µl reaction mixture was used for qPCR (22). qPCR was conducted as follows: 95°C for 30 sec, then 40 cycles of 95°C for 5 sec and 60°C for 30 sec.…”

Section: Methodsmentioning

confidence: 99%

Exosomes derived from mangiferin‑stimulated perivascular adipose tissue ameliorate endothelial dysfunction

et al. 2019

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Perivascular adipose tissue (PVAT) is considered to serve a vital role during the development of endothelial dysfunction. The current study investigated the effect of exosomes derived from mangiferin-stimulated PVAT on endothelial function, including regeneration, migration, apoptosis and inflammation. The number of exosomes secreted by PVAT was increased by stimulation with mangiferin (0.1, 1 or 10 µM), and uptake of these exosomes by endothelial cells was observed. Exosomes produced by stimulation of PVAT with mangiferin reversed the effects of inflammation-induced endothelial dysfunction following palmitic acid (PA) treatment. Furthermore, nuclear factor (NF)-κB signaling in endothelial cells was significantly increased when treated with PA-induced PVAT-derived exosomes, whereas exosomes from the supernatant of PVAT stimulated with mangiferin reduced p65 and p50 phosphorylation levels in the cells, and inhibited p65 transportation to the nucleus. Taken together, the present study demonstrated that exosomes derived from mangiferin-stimulated PVAT supernatant inhibited inflammation-induced endothelial dysfunction via modulation of NF-κB signaling.

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Simultaneous detection of target CNVs and SNVs of thalassemia by multiplex PCR and next‑generation sequencing

Cited by 7 publications

References 44 publications

A Novel Tool for the Analysis and Detection of Copy Number Variants Associated with Haemoglobinopathies

A Novel Tool for the Analysis and Detection of Copy Number Variants Associated with Haemoglobinopathies

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

Exosomes derived from mangiferin‑stimulated perivascular adipose tissue ameliorate endothelial dysfunction

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