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

Abstract

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.

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 the conditions caused by the accumulation of organic acids, Table 3 shows that NIPD was described only for methylmalonic acidemia/aciduria [ 2 , 17 ]. Specifically, the 2022 paper by Lv et al [ 17 ] relied on trios molecular diagnosis performed in 29 cblC type MMA-affected children and their parents by traditional Sanger sequencing.…”

“…Among the conditions caused by the accumulation of organic acids, Table 3 shows that NIPD was described only for methylmalonic acidemia/aciduria [ 2 , 17 ]. Specifically, the 2022 paper by Lv et al [ 17 ] relied on trios molecular diagnosis performed in 29 cblC type MMA-affected children and their parents by traditional Sanger sequencing. In the second pregnancy, invasive prenatal diagnosis (IPD) was performed to determine fetal genotypes and, subsequently, NIPT was performed using a novel MMACHC gene-specific cSMART assay.…”

Non-invasive prenatal diagnosis (NIPD): current and emerging technologies

“…A modified UMI-based NGS approach, validated originally using Sanger sequencing for variants causative of Wilson disease (ATP7B) [89] , has also been developed for NIPD of various monogenic conditions with pathogenic variants causative of autosomal recessive non-syndromic hearing loss (GJB2, GJB3, SLC26A4, RNR1, TRNL1, and COX1) [90,91] , β-thalassaemia (HBB) [92,93] , phenylketonuria (PAH) [94,95] , and methylmalonic acidaemia cb1C type (MMACHC) [96] . This methodology was called circulating single-molecule amplification and resequencing technology (cSMART).…”

Non-invasive prenatal diagnosis (NIPD): how analysis of cell-free DNA in maternal plasma has changed prenatal diagnosis for monogenic disorders

“…These include circulating single-molecule amplification and resequencing technology (cSMART), which involves the introduction of UMI tags, circularization of tagged genomic DNA, inverse PCR, and sequencing. cSMART was developed as a proof-of-concept study for Wilson disease ( ATP7B ) using Sanger sequencing [ 88 ] and, more recently, has employed NGS technologies, showing clinical utility for autosomal recessive non-syndromic hearing loss ( GJB2 and SLC26A4 ) [ 89 , 90 ], β-thalassaemia ( HBB ) [ 91 , 92 ], methylmalonic acidaemia cblC type ( MMACHC ) [ 93 ], and phenylketonuria ( PAH ) [ 94 ]. Other studies have also described the use of barcode-enabled NGS to facilitate molecular counting with single base pair resolution [ 95–97 ], nested PCR and NGS [ 98 ], as well as size-based enrichment of cffDNA prior to diagnostic analysis [ 54 , 97 ].…”