George Koumbaris

Clinical application of whole-genome array CGH during prenatal diagnosis: Study of 25 selected pregnancies with abnormal ultrasound findings or apparently balanced structural aberrations

BackgroundThe purpose of the study was the application and evaluation of array Comparative Genomic Hybridization (array CGH) in selected cases during prenatal diagnosis. Array CGH was applied in 25 fetal samples out of which 15 had normal karyotypes and abnormal ultrasound findings and 10 had apparently balanced structural aberrations with or without abnormal ultrasound findings. DNA was extracted from peripheral blood, chorionic villi samples (CV) and amniotic fluid. Bacterial Artificial Chromosome (BAC) array CGH (Cytochip, BlueGnome Ltd.) of 1 Mb was applied and results were confirmed with either Fluorescence In Situ Hybridization (FISH), Multiplex Ligation-dependant Probe Amplification (MLPA) or Real-Time PCR.ResultsThree out of 25 samples (12%), referred for prenatal array CGH, were found to carry copy number alterations. The number of cases with clinically significant alterations was 2/25 (8%), while one (4%) was of uncertain clinical significance. Two benign Copy Number Variations (CNVs) were also found in 1/25 cases (4%).ConclusionsThe outcome of this study indicates the ability of array CGH to identify chromosomal abnormalities which cannot be detected during routine prenatal cytogenetic analysis, therefore increasing the overall detection rate.

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

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.

Identification of high frequency of Y chromosome deletions in patients with sex chromosome mosaicism and correlation with the clinical phenotype and Y-chromosome instability

A mosaic karyotype consisting of a 45,X cell line and a second cell line containing a normal or an abnormal Y chromosome is relatively common and is associated with a wide spectrum of clinical phenotypes. The aim of this study was to investigate patients with such a mosaic karyotype for Y chromosome material loss and then study the possible association of the absence of these regions with the phenotype, diagnosis, and Y‐chromosome instability. We studied 17 clinically well‐characterized mosaic patients whose karyotype consisted of a 45,X cell line and a second cell line containing a normal or an abnormal Y chromosome. The presence of the Y chromosome centromere was verified by fluorescence in situ hybridization (FISH) and was then characterized by 44 Y‐chromosome specific‐sequence tagged site (STS) markers. This study identifies a high frequency of Yq chromosome deletions (47%). The deletions extend from interval 5 to 7 sharing a common deleted interval (6F), which overlaps with the azoospermia factor region (AZF) region. This study finds no association between Y‐chromosome loci hosting genes other than SRY, and the phenotypic sex, the diagnosis, and the phenotype of the patients. Furthermore, this study shows a possible association of these deletions with Y‐chromosome instability.

Implementation of High Resolution Whole Genome Array CGH in the Prenatal Clinical Setting: Advantages, Challenges, and Review of the Literature

Array Comparative Genomic Hybridization analysis is replacing postnatal chromosomal analysis in cases of intellectual disabilities, and it has been postulated that it might also become the first-tier test in prenatal diagnosis. In this study, array CGH was applied in 64 prenatal samples with whole genome oligonucleotide arrays (BlueGnome, Ltd.) on DNA extracted from chorionic villi, amniotic fluid, foetal blood, and skin samples. Results were confirmed with Fluorescence In Situ Hybridization or Real-Time PCR. Fifty-three cases had normal karyotype and abnormal ultrasound findings, and seven samples had balanced rearrangements, five of which also had ultrasound findings. The value of array CGH in the characterization of previously known aberrations in five samples is also presented. Seventeen out of 64 samples carried copy number alterations giving a detection rate of 26.5%. Ten of these represent benign or variables of unknown significance, giving a diagnostic capacity of the method to be 10.9%. If karyotype is performed the additional diagnostic capacity of the method is 5.1% (3/59). This study indicates the ability of array CGH to identify chromosomal abnormalities which cannot be detected during routine prenatal cytogenetic analysis, therefore increasing the overall detection rate. In addition a thorough review of the literature is presented.

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.

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.

A single gene deletion on 4q28.3: PCDH18--a new candidate gene for intellectual disability?

We report a boy with severe developmental delay, seizures, microcephaly, hypoplastic corpus callosum, internal hydrocephalus and dysmorphic features (narrow forehead, round face, deep-set eyes, blue sclerae, large and prominent ears, nose with anteverted nares, thin upper lip, small and wide-spaced teeth, hyperextensibility of the elbows, wrists, and fingers, fingertip pads, broad hallux, sacral dimple), carrying a 1.53 Mb interstitial deletion at 4q28.3. The deletion was detected by Agilent 105K oligo-array genome hybridization and involves the genomic region between 137,417,338 and 138,947,282 base pairs on chromosome 4 (NCBI build 36). The alteration was inherited from a healthy mother and contains a single gene, PCDH18 which encodes a cadherin-related neuronal receptor thought to play a role in the establishment and function of cell-cell connections in the brain. Thus, haploinsufficiency of this gene may contribute to altered brain development and associated malformations. We found that this deletion is a private inherited copy number variation that is associated with specific clinical findings in our patient and propose the PCDH18 gene as a possible candidate gene for intellectual disability.

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.

A new non-invasive prenatal diagnosis of Down syndrome through epigenetic markers and real-time qPCR

Introduction: Non-invasive prenatal diagnosis (NIPD) of Down syndrome is rapidly evolving. Currently, two applications for NIPD of Down syndrome have been developed with potential and have displayed positive results; the NIPD using next-generation sequencing technologies and the NIPD using the methylated DNA immunoprecipitation (MeDIP) real-time quantitative polymerase chain reaction (qPCR). Areas covered: The MeDIP real-time qPCR approach is based on the identification of differentially methylated regions (DMRs) and their use for discriminating normal from Down syndrome cases. DMRs were identified using high-resolution oligo-arrays. A subgroup of DMRs was selected for further investigation. Through the design of a discriminant equation which combines the results obtained from different DMRs, normal and abnormal cases are correctly classified indicating 100% sensitivity and specificity. Expert opinion: Previous studies have also identified DMRs between non-pregnant female blood and placental DNA. However, these methods have been associated with a number of limitations including the low sensitivity and/or specificity of the assays, the limited number of identified DMRs or methylation sensitive sites and SNPs located on DMRs. These limitations have been overawed by the development of the MeDIP real-time qPCR-based methodology.

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.