Clinical investigational studies for validation of a next-generation sequencing in vitro diagnostic device for cystic fibrosis testing.

PURPOSE: Clinical investigational studies were conducted to demonstrate the accuracy and reproducibility of the Illumina MiSeqDx CF System, a next-generation sequencing (NGS) in vitro diagnostic device for cystic fibrosis testing. METHODS: Two NGS assays - a Clinical Sequencing Assay (Sequencing Assay) and a 139-Variant Assay (Variant Assay) - were evaluated in both an Accuracy Study and a Reproducibility Study, with comparison to bi-directional Sanger sequencing and PCR as reference methods. For each study, positive agreement (PA), negative agreement (NA), and overall agreement (OA) were evaluated. RESULTS: In the Accuracy Study, the Sequencing Assay achieved PA of 99.7% including the polyTG/polyT region and PA of 100% excluding the region. The Variant Assay achieved PA of 100%. NA and OA were >99.99% for both Assays. In the Reproducibility Study, the Sequencing Assay achieved PA of 99.2%; NA and OA were both 99.7%. The Variant Assay achieved PA of 99.8%; NA and OA were both 99.9%. Sample pass rates were 99.7% in both studies for both assays. CONCLUSION: This is the first systematic evaluation of a NGS platform for broad clinical use as an in vitro diagnostic, including accuracy validation with multiple reference methods and reproducibility validation at multiple clinical sites. These NGS-based Assays had accurate and reproducible results which were comparable to or better than other methods currently in clinical use for clinical genetic testing of cystic fibrosis.

DNA sequencing versus standard prenatal aneuploidy screening.

BACKGROUND: In high-risk pregnant women, noninvasive prenatal testing with the use of massively parallel sequencing of maternal plasma cell-free DNA (cfDNA testing) accurately detects fetal autosomal aneuploidy. Its performance in low-risk women is unclear. METHODS: At 21 centers in the United States, we collected blood samples from women with singleton pregnancies who were undergoing standard aneuploidy screening (serum biochemical assays with or without nuchal translucency measurement). We performed massively parallel sequencing in a blinded fashion to determine the chromosome dosage for each sample. The primary end point was a comparison of the false positive rates of detection of fetal trisomies 21 and 18 with the use of standard screening and cfDNA testing. Birth outcomes or karyotypes were the reference standard. RESULTS: The primary series included 1914 women (mean age, 29.6 years) with an eligible sample, a singleton fetus without aneuploidy, results from cfDNA testing, and a risk classification based on standard screening. For trisomies 21 and 18, the false positive rates with cfDNA testing were significantly lower than those with standard screening (0.3% vs. 3.6% for trisomy 21, P<0.001; and 0.2% vs. 0.6% for trisomy 18, P=0.03). The use of cfDNA testing detected all cases of aneuploidy (5 for trisomy 21, 2 for trisomy 18, and 1 for trisomy 13; negative predictive value, 100% [95% confidence interval, 99.8 to 100]). The positive predictive values for cfDNA testing versus standard screening were 45.5% versus 4.2% for trisomy 21 and 40.0% versus 8.3% for trisomy 18. CONCLUSIONS: In a general obstetrical population, prenatal testing with the use of cfDNA had significantly lower false positive rates and higher positive predictive values for detection of trisomies 21 and 18 than standard screening. (Funded by Illumina; ClinicalTrials.gov number, NCT01663350.).

Noninvasive prenatal testing/noninvasive prenatal diagnosis: the position of the National Society of Genetic Counselors.

The 1997 discovery of free fetal DNA in maternal plasma launched clinical researchers' efforts to establish a reliable method for non-invasive prenatal testing for fetal genetic conditions. Various methods, including, but not limited to, massively parallel sequencing (MPS) and selective analysis of cell-free fetal DNA in maternal plasma, have recently been developed as highly sensitive and specific noninvasive screening tools for common fetal chromosome aneuploidies. Incorporating these new noninvasive technologies into clinical practice will impact the current prenatal screening paradigm for fetal aneuploidy, in which genetic counseling plays an integral role. The National Society of Genetic Counselors (NSGC) currently supports Noninvasive Prenatal Testing/Noninvasive Prenatal Diagnosis (NIPT/NIPD) as an option for patients whose pregnancies are considered to be at an increased risk for certain chromosome abnormalities. NSGC urges that NIPT/NIPD only be offered in the context of informed consent, education, and counseling by a qualified provider, such as a certified genetic counselor. Patients whose NIPT/NIPD results are abnormal, or who have other factors suggestive of a chromosome abnormality, should receive genetic counseling and be given the option of standard confirmatory diagnostic testing.

Deletion of hepatocyte nuclear factor-1-beta in an infant with prune belly syndrome.

Prune belly syndrome is a rare congenital disorder characterized by deficiency of abdominal wall muscles, cryptorchidism, and urinary tract anomalies. We have had the opportunity to study a baby with prune belly syndrome associated with an apparently de novo 1.3-megabase interstitial 17q12 microdeletion that includes the hepatocyte nuclear factor-1-beta gene at 17q12. One previous patient, an adult, has been reported with prune belly syndrome and a hepatocyte nuclear factor-1-beta microdeletion. Hepatocyte nuclear factor-1-beta is a widely expressed transcription factor that regulates tissue-specific gene expression and is expressed in numerous tissues including mesonephric duct derivatives, the renal tubule of the metanephros, and the developing prostate of the mouse. Mutations in hepatocyte nuclear factor-1-beta cause the "renal cysts and diabetes syndrome," isolated renal cystic dysplasia, and a variety of other malformations. Based on its expression pattern and the observation of two affected cases, we propose that haploinsufficiency of hepatocyte nuclear factor-1-beta may be causally related to the production of the prune belly syndrome phenotype through a mechanism of prostatic and ureteral hypoplasia that results in severe obstructive uropathy with urinary tract and abdominal distension.

Ultrasound findings in trisomy 22.

We sought to identify the characteristic sonographic findings of fetal trisomy 22 by performing a retrospective review of nine cases of fetal trisomy 22. All cases of chromosomal mosaicism were excluded, as were first-trimester losses. Indications for sonography, gestational age, and sonographically detected fetal anomalies were analyzed. The majority of patients were referred for advanced maternal age or abnormal ultrasound findings on screening exam. Oligohydramnios was the most common sonographic finding, present in 55% of affected fetuses. Intrauterine growth restriction and increased nuchal thickness were slightly less frequent.

Phenotypic characteristics of absent and hypoplastic nasal bones in fetuses with Down syndrome: description by 3-dimensional ultrasonography and clinical significance.

OBJECTIVE: To determine the frequency and clinical significance of bilateral and unilateral hypoplastic nasal bones for the detection of Down syndrome by 3-dimensional ultrasonography. METHODS: Thirty-seven volumes of the fetal skull from fetuses with Down syndrome and 37 from fetuses without abnormalities were analyzed by 1 investigator blinded to fetal karyotype. The maximum intensity projection algorithm was used to reconstruct nasal bones. Ossification patterns were identified in anteroposterior and profile views. Sensitivity, false-positive rates (FPRs), and likelihood ratios (LRs) for detection of Down syndrome were calculated. RESULTS: After exclusions (coronal acquisition [n = 11], hand in front of the face [n = 4], poor imaging [n = 2], incomplete follow-up [n = 2], and anomalies detected after delivery [n = 2]), 53 volumes were analyzed (26 fetuses with Down syndrome and 27 without abnormalities; median gestational age, 21 6/7 weeks [interquartile range, 19 6/7-25 2/7 weeks]). Rendered profile views revealed absent nasal bones in 18.9% (10 of 53) of the fetuses, and, among these, 90% (9 of 10) had Down syndrome (sensitivity, 34.6% [9 of 26]; FPR, 3.7% [1 of 27]; LR, 9.3 [95% confidence interval (CI), 1.3-68.7]). Three ossification patterns were identified in anteroposterior views: (1) normally developed, (2) delayed ossification, and (3) absent nasal bones. Sensitivity, FPR, and LR of absent nasal bones for detecting Down syndrome were 34.6% (9 of 26), 3.7% (1 of 27), and 9.0 (95% CI, 1.3-68.7), respectively. Sensitivity, FPR, and LR of delayed ossification for detecting Down syndrome were 42.3% (11 of 26), 22% (6 of 27), and 1.83 (95% CI, 0.8-4.4). CONCLUSIONS: Absence of nasal bones is associated with the highest risk of Down syndrome. Delayed ossification is associated with a lower risk of Down syndrome than absent nasal bones. These ossification patterns may be indistinguishable on 2-dimensional ultrasonography.