Investigation of Clinically Significant Molecular Aberrations in Patients with Prostate Cancer: Implications for Personalized Treatment, Prognosis and Genetic Testing.

The data on tumor molecular profiling of European patients with prostate cancer is limited. Our aim was to evaluate the prevalence and prognostic and predictive values of gene alterations in unselected patients with prostate cancer. The presence of gene alterations was assessed in patients with histologically confirmed prostate cancer using the ForeSENTIA® Prostate panel (Medicover Genetics), targeting 36 clinically relevant genes and microsatellite instability testing. The primary endpoint was the prevalence of gene alterations in homologous recombination repair (HRR) genes. Overall, 196 patients with prostate cancer were evaluated (median age 72.2 years, metastatic disease in 141 (71.9%) patients). Gene alterations were identified in 120 (61%) patients, while alteration in HRR genes were identified in 34 (17.3%) patients. The most commonly mutated HRR genes were ATM (17, 8.7%), BRCA2 (9, 4.6%) and BRCA1 (4, 2%). The presence of HRR gene alterations was not associated with advanced stage (p = 0.21), age at diagnosis (p = 0.28), Gleason score (p = 0.17) or overall survival (HR 0.72; 95% CI: 0.41-1.26; p = 0.251). We identified clinically relevant somatic gene alterations in European patients with prostate cancer. These molecular alterations have prognostic significance and therapeutic implications and/or may trigger genetic testing in selected patients. In the era of precision medicine, prospective research on the predictive role of these alterations for innovative treatments or their combinations is warranted.

Cell-Free DNA Analysis of Targeted Genomic Regions in Maternal Plasma for Non-Invasive Prenatal Testing of Trisomy 21, Trisomy 18, Trisomy 13, and Fetal Sex.

BACKGROUND: There is great need for the development of highly accurate cost effective technologies that could facilitate the widespread adoption of noninvasive prenatal testing (NIPT). METHODS: We developed an assay based on the targeted analysis of cell-free DNA for the detection of fetal aneuploidies of chromosomes 21, 18, and 13. This method enabled the capture and analysis of selected genomic regions of interest. An advanced fetal fraction estimation and aneuploidy determination algorithm was also developed. This assay allowed for accurate counting and assessment of chromosomal regions of interest. The analytical performance of the assay was evaluated in a blind study of 631 samples derived from pregnancies of at least 10 weeks of gestation that had also undergone invasive testing. RESULTS: Our blind study exhibited 100% diagnostic sensitivity and specificity and correctly classified 52/52 (95% CI, 93.2%-100%) cases of trisomy 21, 16/16 (95% CI, 79.4%-100%) cases of trisomy 18, 5/5 (95% CI, 47.8%-100%) cases of trisomy 13, and 538/538 (95% CI, 99.3%-100%) normal cases. The test also correctly identified fetal sex in all cases (95% CI, 99.4%-100%). One sample failed prespecified assay quality control criteria, and 19 samples were nonreportable because of low fetal fraction. CONCLUSIONS: The extent to which free fetal DNA testing can be applied as a universal screening tool for trisomy 21, 18, and 13 depends mainly on assay accuracy and cost. Cell-free DNA analysis of targeted genomic regions in maternal plasma enables accurate and cost-effective noninvasive fetal aneuploidy detection, which is critical for widespread adoption of NIPT.

Whole-genome fetal and maternal DNA methylation analysis using MeDIP-NGS for the identification of differentially methylated regions.

DNA methylation is an epigenetic marker that has been shown to vary significantly across different tissues. Taking advantage of the methylation differences between placenta-derived cell-free DNA and maternal blood, several groups employed different approaches for the discovery of fetal-specific biomarkers. The aim of this study was to analyse whole-genome fetal and maternal methylomes in order to identify and confirm the presence of differentially methylated regions (DMRs). We have initially utilized methylated DNA immunoprecipitation (MeDIP) and next-generation sequencing (NGS) to identify genome-wide DMRs between chorionic villus sampling (CVS) and female non-pregnant plasma (PL) and peripheral blood (WBF) samples. Next, using specific criteria, 331 fetal-specific DMRs were selected and confirmed in eight CVS, eight WBF and eight PL samples by combining MeDIP and in-solution targeted enrichment followed by NGS. Results showed higher enrichment in CVS samples as compared to both WBF and PL samples, confirming the distinct methylation levels between fetal and maternal DNA for the selected DMRs. We have successfully implemented a novel approach for the discovery and confirmation of a significant number of fetal-specific DMRs by combining for the first time MeDIP and in-solution targeted enrichment followed by NGS. The implementation of this double-enrichment approach is highly efficient and enables the detailed analysis of multiple DMRs by targeted NGS. Also, this is, to our knowledge, the first reported application of MeDIP on plasma samples, which leverages the implementation of our enrichment methodology in the detection of fetal abnormalities in maternal plasma.

FoSTeS, MMBIR and NAHR at the human proximal Xp region and the mechanisms of human Xq isochromosome formation.

The recently described DNA replication-based mechanisms of fork stalling and template switching (FoSTeS) and microhomology-mediated break-induced replication (MMBIR) were previously shown to catalyze complex exonic, genic and genomic rearrangements. By analyzing a large number of isochromosomes of the long arm of chromosome X (i(Xq)), using whole-genome tiling path array comparative genomic hybridization (aCGH), ultra-high resolution targeted aCGH and sequencing, we provide evidence that the FoSTeS and MMBIR mechanisms can generate large-scale gross chromosomal rearrangements leading to the deletion and duplication of entire chromosome arms, thus suggesting an important role for DNA replication-based mechanisms in both the development of genomic disorders and cancer. Furthermore, we elucidate the mechanisms of dicentric i(Xq) (idic(Xq)) formation and show that most idic(Xq) chromosomes result from non-allelic homologous recombination between palindromic low copy repeats and highly homologous palindromic LINE elements. We also show that non-recurrent-breakpoint idic(Xq) chromosomes have microhomology-associated breakpoint junctions and are likely catalyzed by microhomology-mediated replication-dependent recombination mechanisms such as FoSTeS and MMBIR. Finally, we stress the role of the proximal Xp region as a chromosomal rearrangement hotspot.

Molecular profile and clinical features of patients with gliomas using a broad targeted next generation-sequencing panel.

Gliomas are the most common malignant primary brain tumors characterized by poor prognosis. The genotyping of tumors using next generation sequencing (NGS) platforms enables the identification of genetic alterations that constitute diagnostic, prognostic and predictive biomarkers. The present study investigated the molecular profile of 32 tumor samples from 32 patients with high-grade gliomas by implementing a broad 80-gene targeted NGS panel while reporting their clinicopathological characteristics and outcomes. Subsequently, 14 of 32 tumor specimens were also genotyped using a 55-gene NGS panel to validate the diagnostic accuracy and clinical utility of the extended panel. The median follow-up was 19.2 months. In total, 129 genetic alterations including 33 structural variants were identified in 38 distinct genes. Among 96 variants (single nucleotide variants and insertions and deletions), 38 were pathogenic and 58 variants of unknown clinical significance. TP53 was the most frequently mutated gene, followed by PTEN and IDH1 genes. Glioma patients with IDH1 mutant tumors were younger and had significantly longer overall survival compared to patients with wild-type IDH1 tumors. Similarly, tumors with TP53 mutations were more likely observed in younger patients with glioma. Subsequently, a comparison of mutational profiles of samples analyzed by both panels was also performed. Implementation of the comprehensive pan-cancer and the MOL panels resulted in the identification of 37 and 15 variants, respectively. Of those, 13 were common. Comprehensive pan-cancer panel identified 24 additional variants, 22 of which were located in regions that were not targeted by the MOL panel. By contrast, the MOL panel identified two additional variants. Overall, the present study demonstrated that using an extended tumor profile assay instead of a glioma-specific tumor profile panel identified additional genetic changes that may be taken into consideration as potential therapeutic targets for glioma diagnosis and molecular classification.

MeDIP real-time qPCR of maternal peripheral blood reliably identifies trisomy 21.

OBJECTIVE: To reevaluate the efficiency of the 12 differentially methylated regions (DMRs) used in the methylated DNA immunoprecipitation (MeDIP) real-time quantitative polymerase chain reaction (real-time qPCR) based approach, develop an improved version of the diagnostic formula and perform a larger validation study. METHODS: Twelve selected DMRs were checked for copy number variants in the Database of Genomic Variants. The DMRs located within copy number variants were excluded from the analysis. One hundred and seventy-five maternal peripheral blood samples were used to reconstruct and evaluate the new diagnostic formula and for a larger-scale blinded validation study using MeDIP real-time qPCR. RESULTS: Seven DMRs entered the final model of the prediction equation and a larger blinded validation study demonstrated 100% sensitivity and 99.2% specificity. No significant evidence for association was observed between cell free fetal DNA concentration and D value. CONCLUSION: The MeDIP real-time qPCR method for noninvasive prenatal diagnosis of trisomy 21 was confirmed and revalidated in 175 samples with satisfactory results demonstrating that it is accurate and reproducible. We are currently working towards simplification of the method to make it more robust and therefore easily, accurately, and rapidly reproduced and adopted by other laboratories. Nevertheless, larger scale validation studies are necessary before the MeDIP real-time qPCR-based method could be applied in clinical practice.

Proof-of-Concept Pilot Study on Comprehensive Spatiotemporal Intra-Patient Heterogeneity for Colorectal Cancer With Liver Metastasis.

Introduction: The mechanisms underlying high drug resistance and relapse rates after multi-modal treatment in patients with colorectal cancer (CRC) and liver metastasis (LM) remain poorly understood. Objective: We evaluate the potential translational implications of intra-patient heterogeneity (IPH) comprising primary and matched metastatic intratumor heterogeneity (ITH) coupled with circulating tumor DNA (ctDNA) variability. Methods: A total of 122 multi-regional tumor and perioperative liquid biopsies from 18 patients were analyzed via targeted next-generation sequencing (NGS). Results: The proportion of patients with ITH were 53% and 56% in primary CRC and LM respectively, while 35% of patients harbored de novo mutations in LM indicating spatiotemporal tumor evolution and the necessity of multiregional analysis. Among the 56% of patients with alterations in liquid biopsies, de novo mutations in cfDNA were identified in 25% of patients, which were undetectable in both CRC and LM. All 17 patients with driver alterations harbored mutations targetable by molecularly targeted drugs, either approved or currently under evaluation. Conclusion: Our proof-of-concept prospective study provides initial evidence on potential clinical superiority of IPH and warrants the conduction of precision oncology trials to evaluate the clinical utility of I PH-driven matched therapy.

Genomic Epidemiology of the SARS-CoV-2 Epidemic in Cyprus from November 2020 to October 2021: The Passage of Waves of Alpha and Delta Variants of Concern.

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in December 2019 resulted in the coronavirus disease 2019 (COVID-19) pandemic, which has had devastating repercussions for public health. Over the course of this pandemic, the virus has continuously been evolving, resulting in new, more infectious variants that have frequently led to surges of new SARS-CoV-2 infections. In the present study, we performed detailed genetic, phylogenetic, phylodynamic and phylogeographic analyses to examine the SARS-CoV-2 epidemic in Cyprus using 2352 SARS-CoV-2 sequences from infected individuals in Cyprus during November 2020 to October 2021. During this period, a total of 61 different lineages and sublineages were identified, with most falling into three groups: B.1.258 & sublineages, Alpha (B.1.1.7 & Q. sublineages), and Delta (B.1.617.2 & AY. sublineages), each encompassing a set of S gene mutations that primarily confer increased transmissibility as well as immune evasion. Specifically, these lineages were coupled with surges of new infections in Cyprus, resulting in the following: the second wave of SARS-CoV-2 infections in Cyprus, comprising B.1.258 & sublineages, during late autumn 2020/beginning of winter 2021; the third wave, comprising Alpha (B.1.1.7 & Q. sublineages), during spring 2021; and the fourth wave, comprising Delta (B.1.617.2 & AY. sublineages) during summer 2021. Additionally, it was identified that these lineages were primarily imported from and exported to the UK, Greece, and Sweden; many other migration links were also identified, including Switzerland, Denmark, Russia, and Germany. Taken together, the results of this study indicate that the SARS-CoV-2 epidemic in Cyprus was characterized by successive introduction of new lineages from a plethora of countries, resulting in the generation of waves of infection. Overall, this study highlights the importance of investigating the spatiotemporal evolution of the SARS-CoV-2 epidemic in the context of Cyprus, as well as the impact of protective measures placed to mitigate transmission of the virus, providing necessary information to safeguard public health.

A Comprehensive Molecular Epidemiological Analysis of SARS-CoV-2 Infection in Cyprus from April 2020 to January 2021: Evidence of a Highly Polyphyletic and Evolving Epidemic.

The spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) resulted in an extraordinary global public health crisis. In early 2020, Cyprus, among other European countries, was affected by the SARS-CoV-2 epidemic and adopted lockdown measures in March 2020 to limit the initial outbreak on the island. In this study, we performed a comprehensive retrospective molecular epidemiological analysis (genetic, phylogenetic, phylodynamic and phylogeographic analyses) of SARS-CoV-2 isolates in Cyprus from April 2020 to January 2021, covering the first ten months of the SARS-CoV-2 infection epidemic on the island. The primary aim of this study was to assess the transmissibility of SARS-CoV-2 lineages in Cyprus. Whole SARS-CoV-2 genomic sequences were generated from 596 clinical samples (nasopharyngeal swabs) obtained from community-based diagnostic testing centers and hospitalized patients. The phylogenetic analyses revealed a total of 34 different lineages in Cyprus, with B.1.258, B.1.1.29, B.1.177, B.1.2, B.1 and B.1.1.7 (designated a Variant of Concern 202012/01, VOC) being the most prevalent lineages on the island during the study period. Phylodynamic analysis showed a highly dynamic epidemic of SARS-CoV-2 infection, with three consecutive surges characterized by specific lineages (B.1.1.29 from April to June 2020; B.1.258 from September 2020 to January 2021; and B.1.1.7 from December 2020 to January 2021). Genetic analysis of whole SARS-CoV-2 genomic sequences of the aforementioned lineages revealed the presence of mutations within the S protein (L18F, ΔH69/V70, S898F, ΔY144, S162G, A222V, N439K, N501Y, A570D, D614G, P681H, S982A and D1118H) that confer higher transmissibility and/or antibody escape (immune evasion) upon the virus. Phylogeographic analysis indicated that the majority of imports and exports were to and from the United Kingdom (UK), although many other regions/countries were identified (southeastern Asia, southern Europe, eastern Europe, Germany, Italy, Brazil, Chile, the USA, Denmark, the Czech Republic, Slovenia, Finland, Switzerland and Pakistan). Taken together, these findings demonstrate that the SARS-CoV-2 infection epidemic in Cyprus is being maintained by a continuous influx of lineages from many countries, resulting in the establishment of an ever-evolving and polyphyletic virus on the island.

Clinical Significance of Germline Cancer Predisposing Variants in Unselected Patients with Pancreatic Adenocarcinoma.

Our aim was to determine the prevalence, prognostic and predictive role of germline pathogenic/likely pathogenic variants (P/LPVs) in cancer predisposing genes in patients with pancreatic ductal adenocarcinoma (PDAC). Germline testing of 62 cancer susceptibility genes was performed on unselected patients diagnosed from 02/2003 to 01/2020 with PDAC, treated at Hellenic Cooperative Oncology Group (HeCOG)-affiliated Centers. The main endpoints were prevalence of P/LPVs and overall survival (OS). P/LPVs in PDAC-associated and homologous recombination repair (HRR) genes were identified in 22 (4.0%) and 42 (7.7%) of 549 patients, respectively. P/LPVs were identified in 16 genes, including ATM (11, 2.0%) and BRCA2 (6, 1.1%), while 19 patients (3.5%) were heterozygotes for MUTYH P/LPVs and 9 (1.6%) carried the low-risk allele, CHEK2 p.(Ile157Thr). Patients carrying P/LPVs had improved OS compared to non-carriers (22.6 vs. 13.9 months, p = 0.006). In multivariate analysis, there was a trend for improved OS in P/LPV carriers (p = 0.063). The interaction term between platinum exposure and mutational status of HRR genes was not significant (p-value = 0.35). A significant proportion of patients with PDAC carries clinically relevant germline P/LPVs, irrespectively of age, family history or disease stage. The predictive role of these P/LPVs has yet to be defined. ClinicalTrials.gov Identifier: NCT03982446.

Development of a new methylation-based fetal fraction estimation assay using multiplex ddPCR.

BACKGROUND: Non-invasive prenatal testing (NIPT) for fetal aneuploidies has rapidly been incorporated into clinical practice. Current NGS-based methods can reliably detect fetal aneuploidies non-invasively with fetal fraction of at least 4%. Inaccurate fetal fraction assessment can compromise the accuracy of the test as affected samples with low fetal fraction have an increased risk for misdiagnosis. Using a novel set of fetal-specific differentially methylated regions (DMRs) and methylation sensitive restriction digestion (MSRD), we developed a multiplex ddPCR assay for accurate detection of fetal fraction in maternal plasma. METHODS: We initially performed MSRD followed by methylation DNA immunoprecipitation (MeDIP) and NGS on fetal and non-pregnant female tissues to identify fetal-specific DMRs. DMRs with the highest methylation difference between the two tissues were selected for fetal fraction estimation employing MSRD and multiplex ddPCR. Chromosome Y multiplex ddPCR assay (YMM) was used as a reference standard, to develop our fetal fraction estimation model in male pregnancy samples. Additional 123 samples were tested to examine whether the model is sex dependent and/or ploidy dependent. RESULTS: In all, 93 DMRs were identified of which seven were selected for fetal fraction estimation. Statistical analysis resulted in the final model which included four DMRs (FFMM). High correlation with YMM-based fetal fractions was observed using 85 male pregnancies (r = 0.86 95% CI: 0.80-0.91). The model was confirmed using an independent set of 53 male pregnancies. CONCLUSION: By employing a set of well-characterized DMRs, we developed a SNP-, sex- and ploidy-independent methylation-based multiplex ddPCR assay for accurate fetal fraction estimation.

Non-invasive prenatal testing of fetal chromosomal aneuploidies: validation and clinical performance of the veracity test.

INTRODUCTION: Non-Invasive Prenatal Testing (NIPT) for fetal aneuploidies using cell-free DNA (cfDNA) has been widely adopted in clinical practice due to its improved accuracy. A number of NIPT tests have been developed and validated. The purpose of this study is to evaluate the performance of the Veracity NIPT test for sex chromosome aneuploidy (SCA) detection in singleton pregnancies, autosomal aneuploidy detection in twin pregnancies and evaluation of Veracity clinical performance under routine NIPT conditions in a diverse cohort. METHODS: Blinded retrospective study in singleton pregnancies (n = 305); blinded retrospective and prospective study in twin pregnancies (n = 306) and prospective evaluation of clinical performance in singleton and twin pregnancies (n = 10564). RESULTS: Validation study results for the detection of SCAs in singleton pregnancies exhibited 100% sensitivity and specificity and correctly classified 7 (45,X), 4 (47,XXY), 2 (47,XXX) and 1 (47,XYY) cases. Validation study results for autosomal aneuploidy detection in twin pregnancies exhibited 100% sensitivity and specificity and correctly classified 3 trisomy 21, 1 trisomy 18 and 1 trisomy 13 samples. Clinical performance evaluation of Veracity was performed in 10564 pregnancies with median gestational age of 13 weeks, median maternal age 35 years and median gestational weight of 64 kg. Based on confirmation feedback the PPV for trisomies 21, 18 and 13 was estimated at 100% (95% CI, 92-100%), 100% (95% CI, 69-100%) and 71% (95% CI, 29-96%), respectively. Estimated PPV for Monosomy X was 57% (95%CI, 18-90%), while the NPV for SCA detection was estimated at 100% (95% CI, 99.94-100%). CONCLUSION: Veracity NIPT test is based on a very powerful, highly accurate methodology that can be safely applied in the clinical setting.

Targeted capture enrichment followed by NGS: development and validation of a single comprehensive NIPT for chromosomal aneuploidies, microdeletion syndromes and monogenic diseases.

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.

Distal del(4) (q33) syndrome: detailed clinical presentation and molecular description with array-CGH.

The 4q deletion syndrome, comprising all microscopically visible deletions (interstitial and terminal) is a well-recognized distinctive malformation entity, with an estimated incidence of 1:10,000. Here we present the clinical and molecular findings in a 3-year-old male with a de novo distal deletion of 4q33 [46,XY,del(4) (q33)]. Clinical findings of the patient include: hypertelorism, broad nasal bridge, short nose with anteverted nares, long philtrum, thin upper lip, micro-retrognathia, low-set and protruding ears, pre-auricular tag unilaterally, low posterior hairline, clinodactyly of the 5th fingers, tapering fingers, hypospadias, and severe psychomotor retardation. Soon after birth he developed severe hypotonia and feeding difficulties. Echocardiography at 15 months documented aortic supravalvular membrane resulting in mild aortic stenosis and dysplasia of the pulmonary valve. Genome-wide screening using 1 Mb resolution array-CGH and subsequent FISH analyses defined a 18.9-22.9 Mb deletion located at the beginning of 4q33 and extending to the telomere. The description of additional cases with similar distal deletions of 4q33 will allow a more precise prognosis and is therefore of great value for genetic counsellors, while detailed molecular characterization in any well clinically characterized patient is expected to track down individual candidate genes for the specific features of the syndrome.

MeDIP combined with in-solution targeted enrichment followed by NGS: Inter-individual methylation variability of fetal-specific biomarkers and their implementation in a proof of concept study for NIPT.

DNA methylation is the most characterized epigenetic process exhibiting stochastic variation across different tissues and individuals. In non-invasive prenatal testing (NIPT) fetal specific methylated regions can potentially be used as biomarkers for the accurate detection of fetal aneuploidies. The aim of this study was the investigation of inter-individual methylation variability of previously reported fetal-specific markers and their implementation towards the development of a novel NIPT assay for the detection of trisomies 13, 18, and 21. Methylated DNA Immunoprecipitation (MeDIP) combined with in-solution targeted enrichment followed by NGS was performed in 29 CVS and 27 female plasma samples to assess inter-individual methylation variability of 331 fetal-specific differentially methylated regions (DMRs). The same approach was implemented for the NIPT of trisomies 13, 18 and 21 using spiked-in (n = 6) and pregnancy samples (n = 44), including one trisomy 13, one trisomy 18 and four trisomy 21. Despite the variability of DMRs, CVS samples showed statistically significant hypermethylation (p<2e-16) compared to plasma samples. Importantly, our assay correctly classified all euploid and aneuploid cases without any false positive results (n = 44). This work provides the starting point for the development of a NIPT assay based on a robust set of fetal specific biomarkers for the detection of fetal aneuploidies. Furthermore, the assay's targeted nature significantly reduces the analysis cost per sample while providing high read depth at regions of interest increasing significantly its accuracy.

Targeted capture enrichment assay for non-invasive prenatal testing of large and small size sub-chromosomal deletions and duplications.

Noninvasive prenatal testing (NIPT) using whole genome and targeted sequencing has become increasingly accepted for clinical detection of Trisomy 21 and sex chromosome aneuploidies. Few studies have shown that sub-chromosomal deletions or duplications associated with genetic syndromes can also be detected in the fetus noninvasively. There are still limitations on these methodologies such as the detection of variants of unknown clinical significance, high number of false positives, and difficulties to detect small aberrations. We utilized a recently developed targeted sequencing approach for the development of a NIPT assay, for large and small size deletions/duplications, which overcomes these existing limitations. Artificial pregnancies with microdeletion/microduplication syndromes were created by spiking DNA from affected samples into cell free DNA (cfDNA) from non-pregnant samples. Unaffected spiked samples and normal pregnancies were used as controls. Target Capture Sequences (TACS) for seven syndromes were designed and utilized for targeted capture enrichment followed by sequencing. Data was analyzed using a statistical pipeline to identify deletions or duplications on targeted regions. Following the assay development a proof of concept study using 33 normal pregnancies, 21 artificial affected and 17 artificial unaffected pregnancies was carried out to test the sensitivity and specificity of the assay. All 21 abnormal spiked-in samples were correctly classified as subchromosomal aneuploidies while the 33 normal pregnancies or 17 normal spiked-in samples resulted in a false positive result. We have developed an NIPT assay for the detection of sub-chromosomal deletions and duplications using the targeted capture enrichment technology. This assay demonstrates high accuracy, high read depth of the genomic region of interest, and can identify deletions/duplications as small as 0.5 Mb. NIPT of fetal microdeletion/microduplication syndromes can be of enormous benefit in the management of pregnancies at risk both for prospective parents and health care providers.

Identification of a novel 15.5 kb SHOX deletion associated with marked intrafamilial phenotypic variability and analysis of its molecular origin.

Haploinsufficiency of the short stature homeobox contaning SHOX gene has been shown to result in a spectrum of phenotypes ranging from Leri-Weill dyschondrosteosis (LWD) at the more severe end to SHOX-related short stature at the milder end of the spectrum. Most alterations are whole gene deletions, point mutations within the coding region, or microdeletions in its flanking sequences. Here, we present the clinical and molecular data as well as the potential molecular mechanism underlying a novel microdeletion, causing a variable SHOX-related haploinsufficiency disorder in a three-generation family. The phenotype resembles that of LWD in females, in males, however, the phenotypic expression is milder. The 15523-bp SHOX intragenic deletion, encompassing exons 3-6, was initially detected by array-CGH, followed by MLPA analysis. Sequencing of the breakpoints indicated an Alu recombination-mediated deletion (ARMD) as the potential causative mechanism.

The Epigenome View: An Effort towards Non-Invasive Prenatal Diagnosis.

Epigenetic modifications have proven to play a significant role in cancer development, as well as fetal development. Taking advantage of the knowledge acquired during the last decade, great interest has been shown worldwide in deciphering the fetal epigenome towards the development of methylation-based non-invasive prenatal tests (NIPT). In this review, we highlight the different approaches implemented, such as sodium bisulfite conversion, restriction enzyme digestion and methylated DNA immunoprecipitation, for the identification of differentially methylated regions (DMRs) between free fetal DNA found in maternal blood and DNA from maternal blood cells. Furthermore, we evaluate the use of selected DMRs identified towards the development of NIPT for fetal chromosomal aneuploidies. In addition, we perform a comparison analysis, evaluate the performance of each assay and provide a comprehensive discussion on the potential use of different methylation-based technologies in retrieving the fetal methylome, with the aim of further expanding the development of NIPT assays.

Inter-individual methylation variability in differentially methylated regions between maternal whole blood and first trimester CVS.

BACKGROUND: DNA methylation is the most studied form of epigenetic regulation, a process by which chromatin composition and transcription factor binding is altered to influence tissue specific gene expression and differentiation. Such tissue specific methylation patterns are investigated as biomarkers for cancer and cell-free fetal DNA using various methodologies. RESULTS: We have utilized methylation DNA immunoprecipitation (MeDIP) and real-time quantitative PCR to investigate the inter-individual methylation variability of differentially methylated regions (DMRs) on chromosomes 18 and 21. We have characterized 15 newly selected and seven previously validated DMRs in 50, 1(st) trimester Chorionic villus samplings (CVS) and 50 female non-pregnant peripheral blood (WBF) samples. qPCR results from MeDIP and genomic DNA (Input) assays were used to calculate fold enrichment values for each DMR. For all regions tested, enrichment was higher in CVS than in WBF samples with mean enrichments ranging from 0.22 to 6.4 and 0.017 to 1 respectively. Despite inter-individual variability, mean enrichment values for CVS were significantly different than those for WBF in all DMRs tested (p < 0.01). This observation is reinforced by the absence of overlap in CVS and WBF enrichment value distributions for 15 of 22 DMRs. CONCLUSIONS: Our work provides an expansion in the biomarker panel available for non-invasive prenatal diagnosis (NIPD) using the MeDIP-qPCR methology for Down syndrome and can eventually provide the starting point towards the development for assays towards the detection of Edwards syndrome. Furthermore, our data indicate that inter-experimental and inter-individual variation in methylation is apparent, yet the difference in methylation status across tissues is large enough to allow for robust tissue specific methylation identification.