Non-invasive prenatal detection of trisomy 13 using a single nucleotide polymorphism- and informatics-based approach.

PURPOSE:To determine how a single nucleotide polymorphism (SNP)- and informatics-based non-invasive prenatal aneuploidy test performs in detecting trisomy 13. METHODS:Seventeen trisomy 13 and 51 age-matched euploid samples, randomly selected from a larger cohort, were analyzed. Cell-free DNA was isolated from maternal plasma, amplified in a single multiplex polymerase chain reaction assay that interrogated 19,488 SNPs covering chromosomes 13, 18, 21, X, and Y, and sequenced. Analysis and copy number identification involved a Bayesian-based maximum likelihood statistical method that generated chromosome- and sample-specific calculated accuracies. RESULTS:Of the samples that passed a stringent DNA quality threshold (94.1%), the algorithm correctly identified 15/15 trisomy 13 and 49/49 euploid samples, for 320/320 correct copy number calls. CONCLUSIONS:This informatics- and SNP-based method accurately detects trisomy 13-affected fetuses non-invasively and with high calculated accuracy

List of 64 samples that passed quality control, with gestational age, fetal fraction, NATUS-generated copy number result, calculated accuracy, and confirmed karyotype.

<p>For each trisomy 13 case, three confirmed euploid control cases with matching gestational ages (within five days) were blindly selected from a large collection of control cases.</p>a<p>Average calculated accuracy <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096677#pone.0096677-Zimmermann1" target="_blank">[28]</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096677#pone.0096677-SamangoSprouse1" target="_blank">[30]</a> across chromosomes 13, 18, 21, and X.</p>b<p>Karyotype was confirmed by standard invasive diagnostic testing or genetic testing of the cord blood, buccal, saliva, or products of conception. For two samples, confirmed fetal sex chromosome copy number was reported (from a larger, externally blinded cohort) as presence or absence of sex chromosome anomalies (instead of as “XX” or “XY”); NATUS-called karyotypes were identified as correct upon unblinding.</p

Histogram of samples stratified by fetal fraction.

<p>Histogram of samples stratified by fetal fraction.</p

Graphical representation of sequencing data from one maternally-inherited trisomy 13 sample with a 4.2% fetal fraction.

<p>The plot is described as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096677#pone-0096677-g002" target="_blank">Figure 2</a> and in the Results section.</p

Graphical representation of sequencing data from one euploid sample with a 28.1% fetal fraction.

<p>SNPs are assumed to be binary (the algorithm ignores other minor alleles) and are indicated as A and B for simplicity. For each plot, the number of A allele reads is plotted as a fraction of the total allele reads (y-axis) against the linear position of each of several thousand interrogated SNPs on the chromosomes-of-interest (x-axis). The x-axis represents the linear position of each SNP along the indicated chromosome. Interrogated chromosomes are indicated above the plot. Each spot represents a single SNP, where the precise position along the y-axis represents the additive contribution of maternal and fetal cfDNA to the fraction of A allele reads and is thus a function of the sum of fetal and maternal allele reads for that locus as well as of fetal fraction. The contribution of reads from fetal alleles results in distribution of the spots into distinct clusters that can be used to infer chromosomal copy number. Fetal and maternal genotypes at individual SNPs are indicated to the right of the plots. To more easily visualize the maternal and fetal contributions, spots are color-coded according to maternal genotype: SNPs for which the mother is homozygous for the A allele (AA) are indicated in red, those for which the mother is homozygous for the B allele (BB) are indicated in blue, and those for which the mother is heterozygous (AB) are indicated in green. All clusters that are not tightly associated with the limits of the plots are useful for inferring ploidy, as described in the Results section.</p