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

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.

Wilson Hastalığı Olan Gebelerin Obstetrik Sonuçları / Obstetric Outcomes in Pregnant Women with Wilson’s Disease

“…Son zamanlarda, Wilson hastalığı riski taşıyan gebeliklerde fetal genotiplerin tanısında kullanılabilecek noninvaziv bir prenatal test geliştirilmiştir (25). Bizim olgularımızda da her trimesterde serum seruloplazmin, karaciğer ve böbrek fonksiyon testleri, pıhtılaşma profili, tam kan sayımı ve tam idrar tetkiki anne için tekrarlanmıştır.…”

Accessing Genetic Information with Liquid Biopsies

“…In addition to the UMI approach, other error-suppression strategies are also being developed and can be potentially applied to cfDNA to solve the technical shortcomings of conventional PCR on cfDNA. For example, a method dubbed 'circle sequencing' uses CircLigase to circularize denatured single-stranded (ss) DNA fragments, followed by inverse PCR or rolling circle amplification (RCA) ( Figure 2) [38,40]. Unlike conventional PCR, which requires two PCR landing sites flanking the targeted region, inverse PCR primers can be designed adjacent to each other; therefore, it is particularly advantageous in recovering gene fusions with unknown fusion partners ( Figure 2A).…”

“…Currently, to perform prenatal diagnosis on these monogenic disorders, invasive procedures such as chorionic villus sampling or amniocentesis are required. A proof-of-principle study has already shown that monogenic disorders can also be accurately diagnosed prenatally via liquid biopsy [40]. The clinical implementation of NIPD for monogenic disorders will be the next breakthrough of molecular diagnosis in the prenatal health field.…”

Next generation sequencing: Coping with rare genetic diseases in China

“…Haplotype based targeted sequencing of maternal plasma has been shown to be accurate for the diagnosis of HHL (22) and SMA (23). Further, an alternative NIPT method called circulating single molecule amplification and re-sequencing technology (cSMART) has also been demonstrated to accurately genotype the fetus in pregnancies at risk for Wilson Disease (24). In principle, even if there is no knowledge of the causative parental mutations, it is possible to develop these two technologies further for the diagnosis of a broader range of monogenic diseases.…”