Table 1.

Clinical information and noninvasive detection of fetal F8 and F9 sequence variants by ddPCR

Plasma sample*Affected geneSequence variant†Gestation, wkDroplet digital PCR resultsSPRT classificationFetal status§
Total no. of reaction dropletsTotal M allele count‡Total N allele count‡Fetal DNA fraction, %No. of reaction wells to reach SPRT classificationNo. of positive droplets to reach SPRT classification
1A F8 c.6278A>G (p.Asp2093Gly) 18 35 941 192 130 10.9 2 157 Affected Affected 
1B 34 49 337 259 188 21.2 1 77 Affected 
2A F8 c.6278A>G (p.Asp2093Gly) 18 365 097 734 817 3.7 16 1144 Unaffected Unaffected 
2B 36 74 389 221 297 23.5 3 325 Unaffected 
3A F8 c.6532C>T (p.Arg2178Cys) 40 54 396 348 437 24.3 1 127 Unaffected Unaffected 
4A F8 c.6532C>T (p.Arg2178Cys) 24 83 891 252 200 9.8 1 93 Affected Affected 
4B 34 83 535 271 185 15.2 1 92 Affected 
5A F8 c.2417delC (p.Ser806fs) 8 138 458 233 309 4.8 7 243 Unaffected Unaffected 
6A F8 c.1171C>T (p.Arg391Cys) 11 43 266 463 604 4.2 2 502 Unaffected Unaffected 
6B 23 59 215 552 647 8.1 1 212 Unaffected 
7A F8 c.826G>A (p.Val276Met) 28 55 390 289 335 12.2 4 409 Unaffected Unaffected 
8A F9 c.520+13A>G (Splice site) 10 57 734 174 210 17.0 3 250 Unaffected Unaffected 
8B 34 55 860 88 102 25.6 1 28 Unaffected 
9A F9 c.520+13A>G (Splice site) 36 85 166 157 96 33.0 1 58 Affected Affected 
9B 42 85 400 778 610 25.5 1 285 Affected 
10A F9 c.316G>A (p.Gly106Ser) 8 56 146 150 112 0.8 NA NA Unclassified Affected 
10B 23 59 716 193 156 10.0 2 165 Affected 
11A F9 c.874delC (p.Gln292fs) 36 48 538 909 726 17.1 1 374 Affected Affected 
12A F9 c.1144T>C (p.Cys382Arg) 34 63 669 564 434 12.8 1 177 Affected Affected 
12B 38 53 549 3056 2668 10.5 1 843 Affected 
13A F9 c.1069G>A (p.Gly357Arg) 32 58 627 470 636 23.1 1 212 Unaffected Unaffected 
13B 40 38 782 2763 3299 11.9 1 1551 Unaffected 
14A F9 c.802T>A (p.Cys268Ser) 23 50 616 577 505 3.0 4 895 Affected Affected 
14B 32 49 366 138 136 18.4 NA NA Unclassified 
15A F9 c.-35G>A (Promoter, 5′ UTR) 12 140 647 674 671 4.0 NA NA Unclassified Unaffected 
15B 25 134 135 620 784 6.9 6 620 Unaffected 
15C 34 34 347 594 654 5.8 2 578 Unaffected 
Plasma sample*Affected geneSequence variant†Gestation, wkDroplet digital PCR resultsSPRT classificationFetal status§
Total no. of reaction dropletsTotal M allele count‡Total N allele count‡Fetal DNA fraction, %No. of reaction wells to reach SPRT classificationNo. of positive droplets to reach SPRT classification
1A F8 c.6278A>G (p.Asp2093Gly) 18 35 941 192 130 10.9 2 157 Affected Affected 
1B 34 49 337 259 188 21.2 1 77 Affected 
2A F8 c.6278A>G (p.Asp2093Gly) 18 365 097 734 817 3.7 16 1144 Unaffected Unaffected 
2B 36 74 389 221 297 23.5 3 325 Unaffected 
3A F8 c.6532C>T (p.Arg2178Cys) 40 54 396 348 437 24.3 1 127 Unaffected Unaffected 
4A F8 c.6532C>T (p.Arg2178Cys) 24 83 891 252 200 9.8 1 93 Affected Affected 
4B 34 83 535 271 185 15.2 1 92 Affected 
5A F8 c.2417delC (p.Ser806fs) 8 138 458 233 309 4.8 7 243 Unaffected Unaffected 
6A F8 c.1171C>T (p.Arg391Cys) 11 43 266 463 604 4.2 2 502 Unaffected Unaffected 
6B 23 59 215 552 647 8.1 1 212 Unaffected 
7A F8 c.826G>A (p.Val276Met) 28 55 390 289 335 12.2 4 409 Unaffected Unaffected 
8A F9 c.520+13A>G (Splice site) 10 57 734 174 210 17.0 3 250 Unaffected Unaffected 
8B 34 55 860 88 102 25.6 1 28 Unaffected 
9A F9 c.520+13A>G (Splice site) 36 85 166 157 96 33.0 1 58 Affected Affected 
9B 42 85 400 778 610 25.5 1 285 Affected 
10A F9 c.316G>A (p.Gly106Ser) 8 56 146 150 112 0.8 NA NA Unclassified Affected 
10B 23 59 716 193 156 10.0 2 165 Affected 
11A F9 c.874delC (p.Gln292fs) 36 48 538 909 726 17.1 1 374 Affected Affected 
12A F9 c.1144T>C (p.Cys382Arg) 34 63 669 564 434 12.8 1 177 Affected Affected 
12B 38 53 549 3056 2668 10.5 1 843 Affected 
13A F9 c.1069G>A (p.Gly357Arg) 32 58 627 470 636 23.1 1 212 Unaffected Unaffected 
13B 40 38 782 2763 3299 11.9 1 1551 Unaffected 
14A F9 c.802T>A (p.Cys268Ser) 23 50 616 577 505 3.0 4 895 Affected Affected 
14B 32 49 366 138 136 18.4 NA NA Unclassified 
15A F9 c.-35G>A (Promoter, 5′ UTR) 12 140 647 674 671 4.0 NA NA Unclassified Unaffected 
15B 25 134 135 620 784 6.9 6 620 Unaffected 
15C 34 34 347 594 654 5.8 2 578 Unaffected 

ddPCR analyses were performed to test each maternal plasma sample until reaching the fetal genotype classification by the SPRT, or until the maternal plasma was exhausted. Each maternal plasma sample was analyzed by at least 4 reaction wells and evaluated based on the accumulated ddPCR data. The number of reaction wells required for SPRT classification was influenced by the fetal DNA fraction and the total number of allele counts. Total number of positive droplets equals the sum of the M and the N alleles.

NA, not applicable.

*

Maternal blood samples were taken on 1, 2, or 3 occasions from the same pregnancy, denoted as a sample with subscript A, B, or C, respectively.

†

Nomenclature of sequence variants is based on the guidelines of the Human Genome Variation Society.21  GenBank accession NM_000132.3 (F8 mRNA variant 1) and NM_000133.3 (F9 mRNA) were used as the reference sequences for hemophilia A and B sequence variants, respectively.

‡

M allele count, the number of droplets positive for the mutant allele. N allele count, the number of droplets positive for the normal/wild-type allele.

Fetal DNA fractions were determined by the digital ZFY/ZFX assay.

§

Hemophilia status of the newborn was determined from the cord blood sample based on the factor VIII and factor IX assays, as appropriate.

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