Assessment of Fetal Sex Chromosome Aneuploidy Using Directed Cell-Free DNA Analysis (2014)

Prenatal screening for fetal aneuploidies has focused on trisomies 21, 18, and 13. The sex chromosome aneuploidies, monosomy X (45,X), Klinefelter syndrome (47,XXY or 48,XXYY), triple X syndrome (47,XXX), and 47,XYY, with a combined prevalence of 1:500, are more common than the major trisomies. Use of cell-free DNA (cfDNA) analysis in maternal blood allows screening for trisomies 21, 18, and 13 and sex chromosome aneuploidies. Chromosome-selective sequencing of cfDNA, that is, digital analysis of selected regions (DANSR), combined with a fetal-fraction optimized risk of trisomy evaluation (FORTE) algorithm, allows evaluation of chromosomes X and Y. This case-control study was undertaken to determine the clinical performance of chromosome-selective sequencing of cfDNA in maternal blood and the FORTE algorithm for the assessment of fetal sex chromosome aneuploidies. The study included 177 maternal plasma samples from 59 singleton pregnancies with fetal sex chromosome aneuploidies (45,X, n = 49; 47,XXX, n = 6; 47,XXY, n = 1; 47,XYY, n = 3) and 118 with euploid fetuses (46,XY, n = 59; 46,XX, n = 59). All samples were collected before invasive testing for fetal karyotyping at 11 to 13 weeks’ gestation because routine screening indicated an increased risk for fetal trisomies. Digital analysis of selected regions was performed on chromosomes 21, 18, 13, X, and Y, and a FORTE algorithm was used to estimate risk for nondisomic genotypes. A risk cutoff of 1 in 100 for nondisomic genotypes was used to calculate detection and false-positive rates (FPRs). Analysis of cfDNA provided risk scores for 172 (97.2%) of the 177 samples. The median fetal fraction was 12.0% (range, 4.8%–32.0%). Screening by cfDNA analysis provided correct classification in 43 (91.5%) of 47 cases with 45,X, all 5 with 47,XXX, 1 with 47,XXY, and 3 with 47,XYY. In 115 of the 116 euploid pregnancies with results, the classifications were correct, but in 1 case of 46,XX, the classification was 47,XXX, with a risk score of 55 in 100. The distribution of estimated risk for monosomy X by cfDNA analysis in maternal blood from pregnancies with 45,X and euploid fetuses, plotted against fetal fraction, found 39 cases with a risk of greater than 99 in 100 and 1 each with a risk of 1 in 91, 90, 80, 2.8, 1.6, 285, 2500, and 7692 and less than 1 in 10,000. Consequently, 4 (8.5%) of the 47 cases were incorrectly classified as screen negative because the risk was lower than the prespecified cutoff of 1 in 100; 3 were classified as normal female, and 1 was classified as normal male. In the euploid pregnancies, no false-positive cases for monosomy X were found. These results demonstrate the feasibility of chromosome-selective sequencing of cfDNA in maternal blood for assessing the risk for fetal monosomy X and other sex chromosome aneuploidies; 91.5% of cases of 45,X were identified at an FPR of 0%. Whether pregnant women should be screened for these aneuploidies should be further assessed for both individual patient preferences and the clinical utility of such screening. Another factor to consider is the decrease in efficiency of screening because of the likely increase in cumulative FPR.