Noninvasive Prenatal Testing of Methylmalonic Acidemia cblC Type Using the cSMART Assay for MMACHC Gene Mutations

Noninvasive prenatal testing (NIPT) for monogenic disorders has been developed in recent years; however, there are still significant technical and analytical challenges for clinical use. The clinical feasibility of NIPT for methylmalonic acidemia cblC type (cblC type MMA) was investigated using our circulating single-molecule amplification and re-sequencing technology (cSMART). Trios molecular diagnosis was performed in 29 cblC type MMA-affected children and their parents by traditional Sanger sequencing. In the second pregnancy, invasive prenatal diagnosis (IPD) of the pathogenic MMACHC gene was used to determine fetal genotypes, and NIPT was performed using a novel MMACHC gene–specific cSMART assay. Maternal–fetal genotypes were deduced based on the mutation ratio in maternal plasma DNA. Concordance of fetal genotypes between IPD and NIPT, and the sensitivity and specificity of NIPT were determined. After removing two cases with a low P value or reads, the concordance ratio for NIPT and IPD was 100.00% (27/27), and the sensitivity and specificity were 100.00% (54.07–100.00%) and 100.00% (83.89–100.00%), respectively. This study demonstrates that NIPT using the cSMART assay for cblC type MMA was accurate in detecting fetal genotypes. cSMART has a potential clinical application as a prenatal diagnosis and screening tool for carrier and low-risk genotypes of cblC type MMA and other monogenic diseases.

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Noninvasive Prenatal Testing for Wilson Disease by Use of Circulating Single-Molecule Amplification and Resequencing Technology (cSMART)

Clinical Chemistry ◽

10.1373/clinchem.2014.229328 ◽

2015 ◽

Vol 61 (1) ◽

pp. 172-181 ◽

Cited By ~ 51

Author(s):

Weigang Lv ◽

Xianda Wei ◽

Ruolan Guo ◽

Qin Liu ◽

Yu Zheng ◽

...

Keyword(s):

Exome Sequencing ◽

Whole Exome Sequencing ◽

Single Molecule ◽

Wilson Disease ◽

Prenatal Testing ◽

Maternal Plasma ◽

Inverse Pcr ◽

Noninvasive Prenatal Testing ◽

Whole Exome ◽

Monogenic Diseases

Abstract BACKGROUND Noninvasive prenatal testing (NIPT) for monogenic diseases by use of PCR-based strategies requires precise quantification of mutant fetal alleles circulating in the maternal plasma. The study describes the development and validation of a novel assay termed circulating single-molecule amplification and resequencing technology (cSMART) for counting single allelic molecules in plasma. Here we demonstrate the suitability of cSMART for NIPT, with Wilson Disease (WD) as proof of concept. METHODS We used Sanger and whole-exome sequencing to identify familial ATP7B (ATPase, Cu++ transporting, β polypeptide) gene mutations. For cSMART, single molecules were tagged with unique barcodes and circularized, and alleles were targeted and replicated by inverse PCR. The unique single allelic molecules were identified by sequencing and counted, and the percentage of mutant alleles in the original maternal plasma sample was used to determine fetal genotypes. RESULTS Four families with WD pedigrees consented to the study. Using Sanger and whole-exome sequencing, we mapped the pathogenic ATP7B mutations in each pedigree and confirmed the proband's original diagnosis of WD. After validation of cSMART with defined plasma models mimicking fetal inheritance of paternal, maternal, or both parental mutant alleles, we retrospectively showed in second pregnancies that the fetal genotypes assigned by invasive testing and NIPT were concordant. CONCLUSIONS We developed a reliable and accurate NIPT assay that correctly diagnosed the fetal genotypes in 4 pregnancies at risk for WD. This novel technology has potential as a universal strategy for NIPT of other monogenic disorders, since it requires only knowledge of the parental pathogenic mutations.

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Noninvasive Prenatal Diagnosis of Monogenic Diseases by Targeted Massively Parallel Sequencing of Maternal Plasma: Application to β-Thalassemia

Clinical Chemistry ◽

10.1373/clinchem.2012.189589 ◽

2012 ◽

Vol 58 (10) ◽

pp. 1467-1475 ◽

Cited By ~ 112

Author(s):

Kwan-Wood G Lam ◽

Peiyong Jiang ◽

Gary J W Liao ◽

K C Allen Chan ◽

Tak Y Leung ◽

...

Keyword(s):

Prenatal Diagnosis ◽

Massively Parallel Sequencing ◽

Globin Gene ◽

Maternal Plasma ◽

Massively Parallel ◽

Sequencing Data ◽

Plasma Dna ◽

Parallel Sequencing ◽

Noninvasive Prenatal Diagnosis ◽

Monogenic Diseases

Abstract BACKGROUND A genomewide genetic and mutational profile of a fetus was recently determined via deep sequencing of maternal plasma DNA. This technology could have important applications for noninvasive prenatal diagnosis (NIPD) of many monogenic diseases. Relative haplotype dosage (RHDO) analysis, a core step of this procedure, would allow one to elucidate the maternally inherited half of the fetal genome. For clinical applications, the cost and complexity of data analysis might be reduced via targeted application of this approach to selected genomic regions containing disease-causing genes. There is thus a need to explore the feasibility of performing RHDO analysis in a targeted manner. METHODS We performed target enrichment by using solution-phase hybridization followed by massively parallel sequencing of the β-globin gene region in 2 families undergoing prenatal diagnosis for β-thalassemia. We used digital PCR strategies to physically deduce parental haplotypes. Finally, we performed RHDO analysis with target-enriched sequencing data and parental haplotypes to reveal the β-thalassemic status for the fetuses. RESULTS A mean sequencing depth of 206-fold was achieved in the β-globin gene region by targeted sequencing of maternal plasma DNA. RHDO analysis was successful for the sequencing data obtained from the target-enriched samples, including a region in one of the families in which the parents had similar haplotype structures. Data analysis revealed that both fetuses were heterozygous carriers of β-thalassemia. CONCLUSIONS Targeted sequencing of maternal plasma DNA for NIPD of monogenic diseases is feasible.

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Noninvasive Prenatal Testing by Nanopore Sequencing of Maternal Plasma DNA: Feasibility Assessment

Clinical Chemistry ◽

10.1373/clinchem.2015.245076 ◽

2015 ◽

Vol 61 (10) ◽

pp. 1305-1306 ◽

Cited By ~ 21

Author(s):

Suk Hang Cheng ◽

Peiyong Jiang ◽

Kun Sun ◽

Yvonne K Y Cheng ◽

K C Allen Chan ◽

...

Keyword(s):

Prenatal Testing ◽

Maternal Plasma ◽

Nanopore Sequencing ◽

Plasma Dna ◽

Noninvasive Prenatal Testing ◽

Feasibility Assessment

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Universal Haplotype-Based Noninvasive Prenatal Testing for Single Gene Diseases

Clinical Chemistry ◽

10.1373/clinchem.2016.268375 ◽

2017 ◽

Vol 63 (2) ◽

pp. 513-524 ◽

Cited By ~ 54

Author(s):

Winnie W I Hui ◽

Peiyong Jiang ◽

Yu K Tong ◽

Wing-Shan Lee ◽

Yvonne K Y Cheng ◽

...

Keyword(s):

Single Gene ◽

Family Members ◽

Prenatal Testing ◽

Maternal Plasma ◽

Nucleotide Polymorphisms ◽

Plasma Dna ◽

Sequencing Technology ◽

Noninvasive Prenatal Testing ◽

Haplotype Phasing ◽

Mutational Status

Abstract BACKGROUND Researchers have developed approaches for the noninvasive prenatal testing of single gene diseases. One approach that allows for the noninvasive assessment of both maternally and paternally inherited mutations involves the analysis of single nucleotide polymorphisms (SNPs) in maternal plasma DNA with reference to parental haplotype information. In the past, parental haplotypes were resolved by complex experimental methods or inferential approaches, such as through the analysis of DNA from other affected family members. Recently, microfluidics-based linked-read sequencing technology has become available and allows the direct haplotype phasing of the whole genome rapidly. We explored the feasibility of applying this direct haplotyping technology in noninvasive prenatal testing. METHODS We first resolved the haplotypes of parental genomes with the use of linked-read sequencing technology. Then, we identified SNPs within and flanking the genes of interest in maternal plasma DNA by targeted sequencing. Finally, we applied relative haplotype dosage analysis to deduce the mutation inheritance status of the fetus. RESULTS Haplotype phasing and relative haplotype dosage analysis of 12 out of 13 families were successfully achieved. The mutational status of these 12 fetuses was correctly classified. CONCLUSIONS High-throughput linked-read sequencing followed by maternal plasma-based relative haplotype dosage analysis represents a streamlined approach for noninvasive prenatal testing of inherited single gene diseases. The approach bypasses the need for mutation-specific assays and is not dependent on the availability of DNA from other affected family members. Thus, the approach is universally applicable to pregnancies at risk for the inheritance of a single gene disease.

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Single-molecule sequencing reveals a large population of long cell-free DNA molecules in maternal plasma

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2114937118 ◽

2021 ◽

Vol 118 (50) ◽

pp. e2114937118

Author(s):

Stephanie C. Y. Yu ◽

Peiyong Jiang ◽

Wenlei Peng ◽

Suk Hang Cheng ◽

Y. T. Tommy Cheung ◽

...

Keyword(s):

Single Molecule ◽

Dna Analysis ◽

Large Population ◽

Maternal Plasma ◽

Dna Molecules ◽

Plasma Dna ◽

Cell Free Dna ◽

Dna Molecule ◽

Free Dna ◽

Monogenic Diseases

In the field of circulating cell-free DNA, most of the studies have focused on short DNA molecules (e.g., <500 bp). The existence of long cell-free DNA molecules has been poorly explored. In this study, we demonstrated that single-molecule real-time sequencing allowed us to detect and analyze a substantial proportion of long DNA molecules from both fetal and maternal sources in maternal plasma. Such molecules were beyond the size detection limits of short-read sequencing technologies. The proportions of long cell-free DNA molecules in maternal plasma over 500 bp were 15.5%, 19.8%, and 32.3% for the first, second, and third trimesters, respectively. The longest fetal-derived plasma DNA molecule observed was 23,635 bp. Long plasma DNA molecules demonstrated predominance of A or G 5′ fragment ends. Pregnancies with preeclampsia demonstrated a reduction in long maternal plasma DNA molecules, reduced frequencies for selected 5′ 4-mer end motifs ending with G or A, and increased frequencies for selected motifs ending with T or C. Finally, we have developed an approach that employs the analysis of methylation patterns of the series of CpG sites on a long DNA molecule for determining its tissue origin. This approach achieved an area under the curve of 0.88 in differentiating between fetal and maternal plasma DNA molecules, enabling the determination of maternal inheritance and recombination events in the fetal genome. This work opens up potential clinical utilities of long cell-free DNA analysis in maternal plasma including noninvasive prenatal testing of monogenic diseases and detection/monitoring of pregnancy-associated disorders such as preeclampsia.

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