Weigang Lv1, Xianda Wei1, Ruolan Guo1, Qin Liu1, Yu Zheng1, Jiazhen Chang1, Ting Bai1, Haoxian Li1, Jianguang Zhang2, Zhuo Song2, David S Cram3, Desheng Liang4, Lingqian Wu5. 1. State Key Laboratory of Medical Genetics, Central South University, Hunan, China; 2. Berry Genomics, Beijing, China. 3. Berry Genomics, Beijing, China. wulingqian@sklmg.edu.cn liangdesheng@sklmg.edu.cn david.cram@berrygenomics.com. 4. Hunan Jiahui Genetics Hospital, Hunan, China; wulingqian@sklmg.edu.cn liangdesheng@sklmg.edu.cn david.cram@berrygenomics.com. 5. State Key Laboratory of Medical Genetics, Central South University, Hunan, China; wulingqian@sklmg.edu.cn liangdesheng@sklmg.edu.cn david.cram@berrygenomics.com.
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.
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.
Authors: Kezhong Chen; Heng Zhao; Fan Yang; Bengang Hui; Tianyang Wang; Lieu Tu Wang; Yanbin Shi; Jun Wang Journal: BMJ Open Date: 2018-02-06 Impact factor: 2.692