Noninvasive detection of fetal subchromosomal abnormalities by semiconductor sequencing of maternal plasma DNA

Non‐invasive prenatal testing (NIPT) using sequencing of fetal cell‐free DNA from maternal plasma has enabled accurate prenatal diagnosis of aneuploidy and has become increasingly accepted in clinical practice. We investigated whether NIPT using semiconductor sequencing (SSP) could reliably detect subchromosomal deletions/duplications in women carrying high‐risk fetuses. We first demonstrated that increasing concentration of abnormal DNA and sequencing depth improved detection. Subsequently, we analyzed plasma from 1456 pregnant women to develop a method for estimating fetal DNA concentration based on the size distribution of DNA fragments. Finally, we collected plasma from 1476 pregnant women with fetal structural abnormalities detected on ultrasound who also underwent an invasive diagnostic procedure. We used SSP of maternal plasma DNA to detect subchromosomal abnormalities and validated our results with array comparative genomic hybridization (aCGH). With 3.5 million (3.5M) reads, SSP detected 56/78 (71.8%) of subchromosomal abnormalities detected by aCGH. With increased sequencing depth up to 10M reads and restricting the size of abnormalities to more than 1Mb, sensitivity improved to 69/73 (94.5%). Of the 55 false positive samples, 35 were due to deletions/duplications present in maternal DNA, indicating the necessity of a validation test to exclude maternal karyotype abnormalities. This study demonstrates that detection of fetal subchromosomal abnormalities is a viable extension of NIPT based on SSP. Although we focused on the application of cell‐free DNA sequencing for NIPT, we believe that this method has broader applications for genetic diagnosis, such as analysis of circulating tumor DNA for detection of cancer. 1 Comparison of NIPT and aCGH in clinical samplesSample ID dup/del aCGH Result Size (Mp) Syndrome associated with region * NIPT resultLocation predicted by aCGH FC% Location predicted by NIPT 3M reads 5M reads 10M reads 15M readsA0587 del chr16:29673954‐30198600 0.52 16p11.2 microduplicatoin syndrome 17.01 ‐‐ No No No No A0133 dup chr19:327273‐863312 0.54 2.01 ‐‐ No No No No A0553 del chr19:3806228‐4343253 0.54 11.61 ‐‐ No No No No A0519 del chr3:27275‐853692 0.83 3p syndrome 9.57 ‐‐ No No No No A0957 del chr7:157683596‐158602499 0.92 7q36.1‐36.3 Deletion 10.34 ‐‐ No No No No A0281 del chr17:674618‐1859251 1.18 17p13.1 deletion syndrome 9.76 ‐‐ No No No No A1005 dup chr22:22069437‐23318455 1.25 22q11.2 deletion syndrome 19.82 chr22:22‐23 No No Yes Yes A1102 dup chr2:88015932‐89387655 1.37 20.53 chr2:88‐9 Yes Yes Yes Yes A1444 del chr17:72364514‐73777326 1.41 17q25 Duplication 16.93 chr17:71‐73 No No Yes Yes A0187 del chrX:65‐8 1.5 Steroid sulphatase deficiency (STS) NA chrX:5‐7 Yes Yes Yes Yes A0197 dup chrX:65‐8 1.5 Steroid sulphatase deficiency (STS) 9.61 chrX:6‐7 Yes Yes Yes Yes A0403 del chrX:6490000‐8040000 1.55 Steroid sulphatase deficiency (STS) 10.58 chrX:5‐7 Yes Yes Yes Yes A1042 dup chr18:587754‐2180664 1.59 18p11.32‐p11.31 duplication 18.42 chr18:0‐2 No No Yes Yes A1138 dup chr18:36319629‐37915204 1.60 18q12.2‐21.1 deletion 17.27 chr18:35‐37 No No Yes Yes A0495 dup chr16:14408492‐16089759 1.68 16p13.11 microdeletion 20.54 chr16:14‐16 Yes Yes Yes Yes A1285 del chr17:31461588‐33242217 1.78 21.65 chr17:31‐33 No No Yes Yes A0844 dup chr8:1.44E+08‐1.46E+08 1.85 8q22.1‐qter duplication syndrome 10.42 ‐‐ No No No No A1034 del chr4:27770182‐29952659 2.18 14q11‐q22 deletion syndrome 15.77 chr4:27‐29 No No Yes Yes A0248 del chr4:91399112‐93621545 2.22 25.90 chr4:9‐92 Yes Yes Yes Yes A0129 dup chr4:1.57E+08‐1.60E+08 2.42 4q32.1‐q32.2 triplication syndrome 14.98 chr4:157‐159 Yes Yes Yes Yes A0769 del chr22:17299942‐19770514 2.47 DiGeorge syndrome/22q11.2 deletion syndrome NA ‐‐ No No No No A0286 dup chr22:18919942‐21440514 2.52 DiGeorge syndrome/22q11.2 deletion syndrome 13.05 chr22:2‐22 Yes Yes Yes Yes A0686 del chr6:170426‐2753293 2.58 6pter‐p24 deletion syndrome NA chr6:0‐3 No No Yes Yes A0901 del chr13:65054495‐67810584 2.76 28.55 chr13:65‐67 No No Yes Yes A0786 dup chr21:44077514‐46847409 2.77 22.78 chr21:44‐47 No No Yes Yes A0301 del chr2:1.72E+08‐1.75E+08 3 31.85 chr2:171‐174 Yes Yes Yes Yes A0310 del chr22:17096855‐20311763 3.21 DiGeorge syndrome/22q11.2 deletion syndrome 14.74 chr22:26‐3 Yes Yes Yes Yes A0580 dup chr11:1.31E+08‐1.35E+08 3.39 Jacobsen syndrome 8.68 chr11:131‐135 No No Yes Yes A1052 dup chrY:6688691‐10511314 3.82 17.85 chrY:6‐1 Yes Yes Yes Yes A0001 del chr1:814245‐4882747 4.07 1p36 microdeletion syndrome 19.05 chr1:1‐3 Yes Yes Yes Yes A0109 dup chr17:16429920‐20667174 4.24 Smith‐Magenis syndrome 18.88 chr17:16‐19 Yes Yes Yes Yes A0142 del chr22:179‐222 4.3 DiGeorge syndrome/22q11.2 deletion syndrome 11.83 chr22:17‐22 No Yes Yes Yes A1012 del chr1:749625‐5619192 4.87 1p36.33‐36.32 Deletion 14.72 chr1:0‐5 Yes Yes Yes Yes A0844 del chr21:41935392‐46880878 4.95 10.42 chr21:41‐47 No No Yes Yes A0901 dup chrY:17801068‐22916805 5.12 AZEb NA chrY:2‐23 Yes Yes Yes Yes A0433 dup chr15:23076361‐28436403 5.36 15q11.2 deletion syndrome 20.75 chr15:23‐27 Yes Yes Yes Yes A0899 del chr6:1.65E+08‐1.71E+08 5.37 microdeletion 6q27 anosmia 8.93 chr6:164‐171 No Yes Yes Yes A0947 dup chr15:20627802‐26109998 5.48 Prader‐Willi syndrome (type 2) 14.76 chr15:2‐27 Yes Yes Yes Yes A0133 del chr14:1.01E+08‐1.07E+08 6.06 2.01 ‐‐ No No No No A0001 dup chr19:51529057‐59092570 7.56 19.05 chr19:5‐57 Yes Yes Yes Yes A0202 dup chr22:17096855‐25153910 8.06 DiGeorge syndrome/22q11.2 deletion syndrome 24.87 chr22:17‐23 Yes Yes Yes Yes A0894 dup chr3:1.90E+08‐1.99E+08 9.13 3q29 microdeletion syndrome 28.29 chr3:19‐199 Yes Yes Yes Yes A0786 del chr4:61552‐9237101 9.18 Wolf‐Hirschhorn syndrome 22.78 chr4:0‐9 Yes Yes Yes Yes A0107 dup chrY:12571053‐22916805 10.35 AZEa 23.31 chrY:5‐8 Yes Yes Yes Yes A0107 del chrY:1091‐10379571 10.38 sex‐determining region Y/SRY 23.31 chrY:5‐8 Yes Yes Yes Yes A0011 del chr4:61660‐10975146 10.91 Wolf‐Hirschhorn syndrome 20.89 chr4:0‐12 Yes Yes Yes Yes A0256 dup chr12:45001‐11278012 11.23 12p13.33 microdeletion syndrome 19.27 chr12:0‐13 Yes Yes Yes Yes A0578 del chr18:10001‐11497100 11.49 18p deletion syndrome 14.16 chr18:3‐11 Yes Yes Yes Yes A0676 del chr4:1.78E+08‐1.91E+08 12.6 ASD:Small omphalocele. anteriorly placed anus, clef tpalate 8.54 chr4:18‐189 Yes Yes Yes Yes A0248 del chr4:75392078‐88436655 13.04 4q21 deletion syndrome 25.90 chr4:75‐92 Yes Yes Yes Yes A0577 dup chr18:10001‐13279511 13.27 18p deletion syndrome 11.13 chr18:0‐8 Yes Yes Yes Yes A0856 del chr13:1.01E+08‐1.14E+08 13.29 17.84 chr13:101‐113 Yes Yes Yes Yes A0322 dup chr17:87009‐13501809 13.41 17p13.1 deletion syndrome NA chr17:0‐13 Yes Yes Yes Yes A0359 del chr18:142096‐13885315 13.74 18p deletion syndrome 15.81 chr18:0‐13 Yes Yes Yes Yes A0652 dup chr13:99103482‐1.13E+08 14.32 22.09 chr13:5‐95 Yes Yes Yes Yes A1092 dup chr18:4316‐14918854 14.91 18p11.32‐p11.31 duplication 12.48 chr18:0‐15 Yes Yes Yes Yes A0580 del chr15:83800036‐99660791 15.86 15q25 deletion syndrome 8.68 chr15:69‐1 Yes Yes Yes Yes A0310 dup chr11:1.17E+08‐1.35E+08 18.14 Jacobsen syndrome 14.74 chr11:116‐131 Yes Yes Yes Yes A0953 del chr4:61552‐18242617 18.18 4p16.3‐16.1 Deletion 9.45 chr4:0‐18 Yes Yes Yes Yes A0676 dup chr3:93949‐19556862 19.46 3p syndrome 8.54 chr3:0‐2 Yes Yes Yes Yes A0856 dup chr5:204737‐20929211 20.72 Cri du Chat syndrome (5p deletion) 17.84 chr5:7‐21 Yes Yes Yes Yes A0686 dup chr10:1.14E+08‐1.35E+08 21.26 10q26 deletion syndrome NA chr10:113‐133 Yes Yes Yes Yes A0224 dup chr7:1.37E+08‐1.59E+08 22.01 17.61 chr7:135‐157 Yes Yes Yes Yes A1073 dup chr2:161989273‐185124629 23.14 2q31.1 duplication syndrome 9.78 chr2:163‐18 Yes Yes Yes Yes A0202 dup chr7:1.36E+08‐1.59E+08 23.16 24.87 chr7:136‐157 Yes Yes Yes Yes A0359 del chr18:53764221‐77982126 24.22 18q deletion syndrome 15.81 chr18:53‐76 Yes Yes Yes Yes A0302 dup chr13:91‐1.15E+08 25.17 19.03 chr13:91‐113 Yes Yes Yes Yes A0644 del chr13:37474970‐65394654 27.92 13q14 deletion syndrome 14.70 chr13:4‐66 Yes Yes Yes Yes A0224 dup chr13:80744674‐1.13E+08 32.68 17.61 chr13:82‐113 Yes Yes Yes Yes A0894 del chr13:74724554‐1.14E+08 39.35 28.29 chr13:81‐113 Yes Yes Yes Yes A0256 dup chr14:20472548‐64879219 44.41 14q11‐q22 deletion syndrome 19.27 chr14:2‐62 Yes Yes Yes Yes A0519 dup chr18:33381218‐783231 17 44.94 18q deletion syndrome 9.57 chr18:33‐76 Yes Yes Yes Yes A0093 dup chr5:0‐461 46.1 Cri du Chat syndrome (5p deletion) NA chr5:1‐45 Yes Yes Yes Yes A0403 dup chr21:1‐48129895 48.13 Early‐onset Alzheimer disease with cerebral amyloid 10.58 chr21:15‐46 Yes Yes Yes YesangiopathyA0322 del chrX:61529‐48771280 48.71 Xp11.22‐p11.23 microduplication NA chrX:3‐48 Yes Yes Yes Yes A0899 dup chr2:1.90E+08‐2.43E+08 53.11 2q33.1 deletion syndrome 8.93 chr2:188‐241 Yes Yes Yes Yes A0971 del chrX:90374470‐155190083 64.82 Xq26‐28 Deletion 11.03 chrX:93‐15 Yes Yes Yes Yes A0632 dup chr9:261257‐84260002 84 9p deletion syndrome 14.90 chr9:0‐37 Yes Yes Yes Yes* Note: Syndrome is associated with either deletion or duplication in the region but may not match the observed DNA abnormality Dub = Duplication; Del = Deletion;; FC = Fetal DNA Concentraction