Positive predictive value estimates for cell-free noninvasive prenatal screening from data of a large referral genetic diagnostic laboratory.

BACKGROUND: Since its debut in 2011, cell-free fetal DNA screening has undergone rapid expansion with respect to both utilization and coverage. However, conclusive data regarding the clinical validity and utility of this screening tool, both for the originally included common autosomal and sex-chromosomal aneuploidies as well as the more recently added chromosomal microdeletion syndromes, have lagged behind. Thus, there is a continued need to educate clinicians and patients about the current benefits and limitations of this screening tool to inform pre- and posttest counseling, pre/perinatal decision making, and medical risk assessment/management. OBJECTIVE: The objective of this study was to determine the positive predictive value and false-positive rates for different chromosomal abnormalities identified by cell-free fetal DNA screening using a large data set of diagnostic testing results on invasive samples submitted to the laboratory for confirmatory studies. STUDY DESIGN: We tested 712 patient samples sent to our laboratory to confirm a cell-free fetal DNA screening result, indicating high risk for a chromosome abnormality. We compiled data from all cases in which the indication for confirmatory testing was a positive cell-free fetal DNA screen, including the common trisomies, sex chromosomal aneuploidies, microdeletion syndromes, and other large genome-wide copy number abnormalities. Testing modalities included fluorescence in situ hybridization, G-banded karyotype, and/or chromosomal microarray analysis performed on chorionic villus samples, amniotic fluid, or postnatally obtained blood samples. Positive predictive values and false-positive rates were calculated from tabulated data. RESULTS: The positive predictive values for trisomy 13, 18, and 21 were consistent with previous reports at 45%, 76%, and 84%, respectively. For the microdeletion syndrome regions, positive predictive values ranged from 0% for detection of Cri-du-Chat syndrome and Prader-Willi/Angelman syndrome to 14% for 1p36 deletion syndrome and 21% for 22q11.2 deletion syndrome. Detection of sex chromosomal aneuploidies had positive predictive values of 26% for monosomy X, 50% for 47,XXX, and 86% for 47,XXY. CONCLUSION: The positive predictive values for detection of common autosomal and sex chromosomal aneuploidies by cell-free fetal DNA screening were comparable with other studies. Identification of microdeletions was associated with lower positive predictive values and higher false-positive rates, likely because of the low prevalence of the individual targeted microdeletion syndromes in the general population. Although the obtained positive predictive values compare favorably with those seen in traditional screening approaches for common aneuploidies, they highlight the importance of educating clinicians and patients on the limitations of cell-free fetal DNA screening tests. Improvement of the cell-free fetal DNA screening technology and continued monitoring of its performance after introduction into clinical practice will be important to fully establish its clinical utility. Nonetheless, our data provide valuable information that may aid result interpretation, patient counseling, and clinical decision making/management.

Detection of fetal subchromosomal abnormalities by sequencing circulating cell-free DNA from maternal plasma.

BACKGROUND: The development of sequencing-based noninvasive prenatal testing (NIPT) has been largely focused on whole-chromosome aneuploidies (chromosomes 13, 18, 21, X, and Y). Collectively, they account for only 30% of all live births with a chromosome abnormality. Various structural chromosome changes, such as microdeletion/microduplication (MD) syndromes are more common but more challenging to detect. Recently, several publications have shown results on noninvasive detection of MDs by deep sequencing. These approaches demonstrated the proof of concept but are not economically feasible for large-scale clinical applications. METHODS: We present a novel approach that uses low-coverage whole genome sequencing (approximately 0.2×) to detect MDs genome wide without requiring prior knowledge of the event's location. We developed a normalization method to reduce sequencing noise. We then applied a statistical method to search for consistently increased or decreased regions. A decision tree was used to differentiate whole-chromosome events from MDs. RESULTS: We demonstrated via a simulation study that the sensitivity difference between our method and the theoretical limit was <5% for MDs ≥9 Mb. We tested the performance in a blinded study in which the MDs ranged from 3 to 40 Mb. In this study, our algorithm correctly identified 17 of 18 cases with MDs and 156 of 157 unaffected cases. CONCLUSIONS: The limit of detection for any given MD syndrome is constrained by 4 factors: fetal fraction, MD size, coverage, and biological and technical variability of the event region. Our algorithm takes these factors into account and achieved 94.4% sensitivity and 99.4% specificity.

Telomerase reverse transcriptase haploinsufficiency and telomere length in individuals with 5p- syndrome.

Telomerase, which maintains the ends of chromosomes, consists of two core components, the telomerase reverse transcriptase (TERT) and the telomerase RNA (TERC). Haploinsufficiency for TERC or TERT leads to progressive telomere shortening and autosomal dominant dyskeratosis congenita (DC). The clinical manifestations of autosomal dominant DC are thought to occur when telomeres become critically short, but the rate of telomere shortening in this condition is unknown. Here, we investigated the consequences of de novo TERT gene deletions in a large cohort of individuals with 5p- syndrome. The study group included 41 individuals in which the chromosome deletion resulted in loss of one copy of the TERT gene at 5p15.33. Telomere length in peripheral blood cells from these individuals, although within the normal range, was on average shorter than in normal controls. The shortening was more significant in older individuals suggesting an accelerated age-dependent shortening. In contrast, individuals with autosomal dominant DC due to an inherited TERC gene deletion had very short telomeres, and the telomeres were equally short regardless of the age. Although some individuals with 5p- syndrome showed clinical features that were reminiscent of autosomal dominant DC, these features did not correlate with telomere length, suggesting that these were not caused by critically short telomeres. We conclude that a TERT gene deletion leads to slightly shorter telomeres within one generation. However, our results suggest that several generations of TERT haploinsufficiency are needed to produce the very short telomeres seen in patients with DC.

Prenatal diagnosis of 5p deletion syndrome following abnormally low maternal serum human chorionic gonadotrophin.

Prenatal diagnosis of 5p deletion syndrome, or cri du chat, following an abnormally low measurement of a screening of serum human chorionic gonadotrophin (hCG), is reported. Karyotyping following amniocentesis revealed a terminal deletion in the short arm of one chromosome 5. The pregnancy was electively terminated. 5p deletion syndrome has been described with abnormally high hCG levels and normal hCG levels. This is the first report of its association with abnormally low levels. The association between chromosomal abnormalities and hCG is discussed.

FISH analysis of terminal deletions in patients diagnosed with cri-du-chat syndrome.

Most patients with cri-du-chat syndrome have a de novo deletion of the short arm of chromosome 5 (5p). In order to perform extensive phenotype-genotype correlation studies, a relatively easy method for the precise determination of the extent of a patient's deletion is essential. Towards this purpose, a set of minimally overlapping YAC clones that span 5p was identified. A BAC that maps at or near the 5p telomere was also used. A total of 110 patients with previously determined de novo terminal deletions by standard cytogenetic approaches were reanalyzed using the YAC clones and fluorescent in situ hybridization (FISH). Of the 110 samples, 4 patients were determined to have interstitial deletions, 1 patient had an unbalanced translocation, and no deletion could be detected in 2 patients. The FISH results in the 7 patients affect the clinical prognosis for some of these patients. These results demonstrate the need for supplementing standard cytogenetics with FISH analysis when an abnormal karyotype is detected.