Targeted capture enrichment followed by NGS: development and validation of a single comprehensive NIPT for chromosomal aneuploidies, microdeletion syndromes and monogenic diseases
George Koumbaris1, Achilleas Achilleos1, Michalis Nicolaou1, Charalambos Loizides1, Kyriakos Tsangaras1, Elena Kypri1, Petros Mina1, Carolina Sismani2,3, Voula Velissariou1,4, Georgia Christopoulou5, Pantelis Constantoulakis5, Emmanouil Manolakos6, Ioannis Papoulidis6, Danai Stambouli7, Marios Ioannides1 and Philippos Patsalis1*
Background: Non-invasive prenatal testing (NIPT) has been widely adopted for the detection of fetal aneuploidies and microdeletion syndromes, nevertheless, limited clinical utilization has been reported for the non-invasive prenatal screening of monogenic diseases. In this study, we present the development and validation of a single comprehensive NIPT for prenatal screening of chromosomal aneuploidies, microdeletions and 50 autosomal recessive disorders associated with severe or moderate clinical phenotype.
Results: We employed a targeted capture enrichment technology powered by custom TArget Capture Sequences (TACS) and multi-engine bioinformatics analysis pipeline to develop and validate a novel NIPT test. This test was validated using 2033 cell-fee DNA (cfDNA) samples from maternal plasma of pregnant women referred for NIPT and paternal genomic DNA. Additionally, 200 amniotic fluid and CVS samples were used for validation purposes. All NIPT samples were correctly classified exhibiting 100% sensitivity (CI 89.7–100%) and 100% specificity (CI 99.8–100%) for chromosomal aneuploidies and microdeletions. Furthermore, 613 targeted causative mutations, of which 87 were unique, corresponding to 21 monogenic diseases, were identified. For the validation of the assay for prenatal diagnosis purposes, all aneuploidies, microdeletions and point mutations were correctly detected in all 200 amniotic fluid and CVS samples.
Conclusions: We present a NIPT for aneuploidies, microdeletions, and monogenic disorders. To our knowledge this is the first time that such a comprehensive NIPT is available for clinical implementation.
Keywords: NIPT, Monogenic diseases, Cell-free DNA, Aneuploidies, Microdeletions
Until recently, prenatal screening for fetal aneuploidies relied on the measurement of maternal serum biochem- ical markers combined with fetal ultrasound markers. The discovery of cell-free fetal DNA (cffDNA) in mater- nal circulation prompted the development of non- invasive prenatal testing (NIPT), opening a new era in prenatal screening . Since its introduction, different methods have been applied for the detection of fetal aneuploidies, mainly employing whole genome or tar- geted approaches combined with Next Generation Se- quencing (NGS). The success of such methods has been highlighted in several clinical validation studies that demonstrate the ability for high aneuploidy detection rates [2–9]. This has led to the endorsement of NIPT by several professional bodies as a primary screening method regardless of the pregnancy risk status [10–12]. Furthermore, in the absence of specific markers, NIPT can be used for sex chromosomal aneuploidies and for select microdeletion syndromes .
For the non-invasive prenatal screening of monogenic diseases limited clinical utilization has been reported. Early studies were focused on disease detection or exclu- sion based on the presence of de novo or paternally inherited mutations respectively, using PCR approaches, allowing only for the screening of a limited number of monogenic diseases [13–15].
Recently, the advent of high precision and high throughput technologies such as NGS and digital PCR, catalyzed the development of higher sensitivity assays for the detection of monogenic diseases in cfDNA [16–21], most of them requiring the parental haplotype to interpret the fetal inheritance pat- tern [18, 22, 23]. Nevertheless, limited scalability, high cost and increased complexity in assay performance and data analysis rendered their clinical implementation challenging . Recently, Luo et al. described in a proof of concept study the feasibility of integrating three tests in a single NIPT for the detection of aneuploidies, large copy num- ber variants (> 20 Mb) and a limited number of single gene diseases using target capture enrichment. The au- thors employed a direct NIPT approach for screening of monogenic diseases, which is highly dependent on fetal fraction. Despite the promising results, the sensitivity of the assay was low, especially for the detection of point mutations in the cffDNA even in samples with high fetal fraction. As the authors conclude, additional techno- logical optimizations are needed to increase the test’s ac- curacy and a larger sample cohort is required for validation and determination of the analytical perform- ance of the test prior to clinical implementation . For the determination of the fetal risk for monogenic diseases, unlike Luo et al., we followed a conventional, fetal fraction independent prenatal screening approach by assessing the fetal risk based on the parental carrier status, in the same workflow as cffDNA analysis for an- euploidies and microdeletions.
Addressing the need of NIPT as prenatal screening for both chromosomal and monogenic diseases, we have de- veloped and validated a new, single, comprehensive NIPT providing the fetal risk for these types of genetic conditions. By employing our existing hybrid capture- based technology with minor modifications  and multi-engine bioinformatics analysis pipeline, we present the development and validation of a comprehensive NIPT which offers prenatal screening for aneuploidies of chromosomes 13, 18, 21, X, Y, four microdeletion syn- dromes and 50 autosomal recessive monogenic disorders with severe or moderate clinical phenotype. The fetal risk for aneuploidies and microdeletions is provided based on the analysis of cffDNA present in maternal plasma, whilst the fetal risk for the autosomal recessive monogenic diseases is determined based on Mendelian law of inheritance by combining maternal and paternal carrier status information using cell free maternal DNA (cfmDNA) and paternal DNA respectively.
Results The workflow of this novel comprehensive NIPT pre- natal screening test for aneuploidies of 13, 18, 21, X, Y, four microdeletion syndromes and 50 single gene diseases, followed by prenatal diagnosis for these disor- ders in case of high risk pregnancy is described in Additional file 1. The cfDNA, consisting of cffDNA and cfmDNA, from pregnant women of at least 10 weeks of gestation and paternal genomic DNA (n = 2033), were simultaneously analyzed for the detection of chromosomal aneuploidies (trisomy 21, trisomy 18 and trisomy 13), sex chromosome aneuploidies (SCA) (monosomy X, Klinefelter syndrome, trisomy X, XYY, and XXYY), four microdeletion syndromes (1p36 deletion syndrome, Wolf-Hirschhorn syn- drome, Smith-Magenis syndrome and 22q11.2 deletion syndrome) and point mutations for 50 autosomal recessive conditions (Additional file 2). In the 2033 samples analyzed with this single com- prehensive NIPT, we identified all samples correctly, exhibiting 100% sensitivity (CI 89.7–100%) and 100% specificity (CI 99.8–100%) for chromosomal aneu- ploidies and microdeletion syndromes. Specifically, 22/ 22 trisomy 21, 4/4 trisomy 18, 1/1 trisomy 13 cases, 2/2 SCAs and 5/5 microdeletion syndromes (three cases of 22q11.2 deletion, one case of Wolf–Hirschhorn syn- drome and one case of Smith-Magenis syndrome) were correctly classified (Table 1). The results were in agreement with the invasive pre- natal diagnosis results performed by the collaborating laboratories for all tested samples.
PRENATAL, POSTNATAL ve ONKOLOJİ testlerinde bizi tercih etmeniz için yüzlerce neden sıralayabiliriz.Ama en önemli sebep yüksek doğruluk oranımız ve hızlı test sonuçlandırmamızdır.Hastalar ve Klinisyenler için bizi neden tercih edeceğinize dair verileri listeledik.Aşağıdaki linkten içeriğe ulaşabilirsiniz.
VERACITY ; Kopya Varyantları (CNV), tekrarlayan DNA elemanları ve karmaşık genomik yapılardan kaçınmak için, özel olarak geliştirilmiş, uluslararası akredite edilmiş hedef yakalama dizilerini (TACS) kullanır. Bu hedefe yönelik yaklaşım diğer non-invaziv prenatal tarama testerinde olan sorunların giderilmesini sağlayarak VERACITY nin duyarlılığını , güvenilirliğini ve doğruluğunu artırır.
Okuma derinliği , analiz esnasında genomdaki nükleotidlerin kaç kez okunduğudur.VERACITY , hedeflenen bölgelerden ilgili kromozomların DNA parçacıkları yakalar .Böylece , bu seçilen bölgeleri son derece yüksek okuma derinliğinde okuyabilmektedir ve bu analizin istatistiksel doğruluğunu yükseltir ve VERACITYnin hassasiyetini ve özgüllüğünü artırır.
VERACITY, güvenilir şekilde fetal cfDNA ve maternal cfDNAyı ayırt etmek için bilgilendirici lokusları kullanır.Tescilli özel bir biyoinformatik yazılım, fetal fraksiyonu hesaplamak için bu bilgilendirici lokusları yüksek okuma derinliği sayımlarını kullanır.Doğru fetal kesir ölçümleri VERACITYnin sağlamlığını ve güvenilirliğini artırır.
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