One-step noninvasive prenatal testing (NIPT) for autosomal recessive homozygous point mutations using digital PCR

Chang, Mun Seog; Ahn, Soyeon; Kim, Min Young; Han, Jin Hee; Park, Hye Rim; Seo, Han Kyu; Yoon, Jinsun; Lee, Seungmin; Oh, Doo Yi; Kang, Changsoo; Choi, Byung Yoon

doi:10.1038/s41598-018-21236-w

Cited by 33 publications

(22 citation statements)

References 29 publications

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“…[6][7][8][9] Both low throughput methods (eg, real-time PCR) and high throughput technologies (eg, Next-Generation Sequencing [NGS]) have been tested on cffDNA with different performances. [10][11][12][13] It is believed that detection of paternal mutations with NGS can be readily achieved with high sensitivity and specificity, 14 while detection of maternal allele in cffDNA is more challenging mainly due to maternal interference. Thus, a new generation of noninvasive prenatal testing/diagnosis methods, which can accurately detect diverse genetic abnormalities, are needed for pregnant women from families with risks of monogenic disorders.…”

Section: Introductionmentioning

confidence: 99%

A novel method for noninvasive diagnosis of monogenic diseases from circulating fetal cells

Chang

Zhu

et al. 2020

Prenatal Diagnosis

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Objective: To establish a method for noninvasive fetal cell isolation from maternal blood and prenatal testing of monogenic diseases by a combination of direct sequencing and targeted NGS-based SNP haplotyping from single fetal cells. Method: Peripheral blood of pregnant women in two families (congenital deafness and ichthyosis) was collected. After density-based separation and immunostaining with multiple biomarkers, candidate fetal cells were identified by high-throughput imagine analysis and picked up by automation. Individual fetal cells were subjected to STR-genotyping to identify their origin. Pathogenic mutations were identified by direct Sanger sequencing, and a combination of targeted NGS and SNP haplotyping using a custom panel. All the results were compared with amniotic fluid DNA. Results: Fetal trophoblasts were successfully harvested from maternal blood. STRgenotyping confirmed the fetal origin. Direct sequencing of pathogenic genetic mutations in fetal cells showed consistent results with amniotic fluid samples. For congenital deafness family, NGS-based SNP haplotyping also correctly identified the fetal haplotype. This single cell haplotyping method can be used to diagnose various genetic diseases. Conclusion: We have established a method for noninvasive prenatal testing of monogenic diseases from circulating trophoblast cells. This cell-based NIPT can be further applied to the prenatal diagnosis of various monogenic diseases.

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Section: Introductionmentioning

confidence: 99%

A novel method for noninvasive diagnosis of monogenic diseases from circulating fetal cells

Chang

Zhu

et al. 2020

Prenatal Diagnosis

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“…This makes it difficult to evaluate the clinical performance, resulting in large ranges of confidence intervals and low sensitivity compared to other studies. 39,40 Although our method was less complicated comparing with previous methods, 41,42 our method could not provide a full fetal genotype interpretation, and consequently was only suitable for analyzing de novo mutations and paternal alleles. Nonetheless, as a proofof-concept study, the data collected from 68 pregnancies demonstrated the stability and repeatability of TAGs-seq in simultaneously generating low-pass whole-genome data and high-depth data of target genes.…”

Section: Discussionmentioning

confidence: 98%

Simultaneous detection of fetal aneuploidy, de novoFGFR3 mutations and paternally derived β‐thalassemia by a novel method of noninvasive prenatal testing

Yang

et al. 2021

Prenatal Diagnosis

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Objective: The aim is to develop a novel noninvasive prenatal testing (NIPT) method that simultaneously performs fetal aneuploidy screening and the detection of de novo and paternally derived mutations. Methods: A total of 68 pregnancies, including 26 normal pregnancies, 7 cases with fetal aneuploidies, 7 cases with fetal achondroplasia or thanatophoric dysplasia, 18 cases with fetal skeletal abnormalities, and 10 cases with β-thalassemia high risk were recruited. Plasma cell-free DNA was amplified by Targeted And Genome-wide simultaneous sequencing (TAGs-seq) to generate around 99% of total reads covering the whole-genome region and around 1% covering the target genes. The reads on the whole-genome region were analyzed for fetal aneuploidy using a binary hypothesis T-score and the reads on target genes were analyzed for point mutations by calculating the minor allelic frequency of loci on FGFR3 and HBB. TAGs-seq results were compared with conventional NIPT and diagnostic results. Results: In each sample, TAGs-seq generated 44.7-54 million sequencing reads covering the whole-genome region of 0.1-3� and the target genes of >1000�depth. All cases of fetal aneuploidy and de novo mutations of achondroplasia/thanatophoric dysplasia were identified with high sensitivities and specificities except for one false-negative paternal mutation of β-thalassemia. Conclusions: TAGs-seq is a novel NIPT method that combines the fetal aneuploidy screening and the detection of de novo FGFR3 mutations and paternal HBB mutations. Lin Yang and Yujing Wu joint first authors. This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made.

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“…Some of these methods, namely, droplet digital PCR, may be available in the near future for use in noninvasive prenatal testing. 23,24…”

Section: Discussionmentioning

confidence: 99%

Noninvasive prenatal screening test for compound heterozygous beta thalassemia using an amplification refractory mutation system real-time polymerase chain reaction technique

Suwannakhon¹,

Pangeson²,

Seeratanachot³

et al. 2019

Hematol Rep

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We propose using a modified amplification refractory mutation system real-time polymerase chain reaction (ARMS RTPCR) technique to exclude the invasive prenatal diagnosis for a non-paternally inherited beta thalassemia mutation in couples atrisk for having a baby with CHBT. The ARMS RT-PCR method was performed for 36 at-risk couples by using isolated fetal cell-free DNA from maternal plasma. The modified ARMS RT-PCR primers targeted one of the following paternally inherited beta thalassemia mutation: -28 A→G, CD17 A→T, CD 26 G→A, IVS1-1 G→T and CD 41-42 -CTTT. The method could be successfully employed for NIPST starting with the 7th week of gestation. The results showed that 19 pregnant women were negative for PIBTM (53%). After an on-track and on-time of one year, including postnatal thalassemia blood tests, none of the babies showed symptoms or signs of beta thalassemia disease. We concluded that the modified ARMS RT-PCR method was an accurate, cost-effective and feasible method for use as a NIPST for at-risk couples with the potential of having a baby with CHBT.

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One-step noninvasive prenatal testing (NIPT) for autosomal recessive homozygous point mutations using digital PCR

Cited by 33 publications

References 29 publications

A novel method for noninvasive diagnosis of monogenic diseases from circulating fetal cells

A novel method for noninvasive diagnosis of monogenic diseases from circulating fetal cells

Simultaneous detection of fetal aneuploidy, de novoFGFR3 mutations and paternally derived β‐thalassemia by a novel method of noninvasive prenatal testing

Noninvasive prenatal screening test for compound heterozygous beta thalassemia using an amplification refractory mutation system real-time polymerase chain reaction technique

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