Noninvasive prenatal testing for Wilson disease by use of circulating single-molecule amplification and resequencing technology (cSMART).
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.
State Key Laboratory of Medical Genetics, Central South University, Hunan, China;
Weigang
W
Lv
State Key Laboratory of Medical Genetics, Central South University, Hunan, China;
Xianda
X
Wei
State Key Laboratory of Medical Genetics, Central South University, Hunan, China;
Ruolan
R
Guo
State Key Laboratory of Medical Genetics, Central South University, Hunan, China;
Qin
Q
Liu
State Key Laboratory of Medical Genetics, Central South University, Hunan, China;
Yu
Y
Zheng
State Key Laboratory of Medical Genetics, Central South University, Hunan, China;
Jiazhen
J
Chang
State Key Laboratory of Medical Genetics, Central South University, Hunan, China;
Ting
T
Bai
State Key Laboratory of Medical Genetics, Central South University, Hunan, China;
Haoxian
H
Li
Berry Genomics, Beijing, China.
Jianguang
J
Zhang
Berry Genomics, Beijing, China.
Zhuo
Z
Song
Berry Genomics, Beijing, China. wulingqian@sklmg.edu.cn liangdesheng@sklmg.edu.cn david.cram@berrygenomics.com.
David S
DS
Cram
Hunan Jiahui Genetics Hospital, Hunan, China; wulingqian@sklmg.edu.cn liangdesheng@sklmg.edu.cn david.cram@berrygenomics.com.
Desheng
D
Liang
State Key Laboratory of Medical Genetics, Central South University, Hunan, China; wulingqian@sklmg.edu.cn liangdesheng@sklmg.edu.cn david.cram@berrygenomics.com.
Lingqian
L
Wu
D000251:Adenosine Triphosphatases / Q000235:genetics; D000483:Alleles; D027682:Cation Transport Proteins / Q000235:genetics; D000073840:Copper-Transporting ATPases; D004247:DNA / Q000097:blood / Q000235:genetics; D004252:DNA Mutational Analysis / Q000379:methods*; D015342:DNA Probes; D005260:Female; D005865:Gestational Age; D006527:Hepatolenticular Degeneration / Q000097:blood* / Q000196:embryology / Q000235:genetics*; D006579:Heterozygote; D006720:Homozygote; D006801:Humans; D008297:Male; D025202:Molecular Diagnostic Techniques / Q000295:instrumentation / Q000379:methods*; D011247:Pregnancy; D011296:Prenatal Diagnosis / Q000295:instrumentation / Q000379:methods*
D027682:Cation Transport Proteins; D015342:DNA Probes; D004247:DNA; D000251:Adenosine Triphosphatases; C000612464:ATP7B protein, human; D000073840:Copper-Transporting ATPases