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.

Phenotype-genotype correlation of a patient with a "balanced" translocation 9;15 and cryptic 9q34 duplication and 15q21q25 deletion.

We report on a 2-year-old boy with intellectual disabilities, distinctive facies, hypotonia, cardiac, and renal malformations. During his infancy he had recurrent episodes of apnea, cyanosis, and bradycardia. Chromosomal analysis showed a de novo apparently balanced translocation 46,XY,t(9;15)(q31;q26)dn. The use of array-comparative genomic hybridization (CGH) however, revealed the presence of additional cryptic complex chromosomal rearrangements involving a approximately 5-5.8 Mb distal duplication on chromosome 9 (9q34.1 --> 9q34.3), and deletions on three separate regions of chromosome 15 adding to approximately 8.1-12.2 Mb (15q21.2 --> 15q21.3, 15q22.31 --> 15q23, 15q25.1 --> 15q25.2). During confirmation with fluorescence in situ hybridization (FISH) an inversion was unexpectedly revealed on chromosome 15 (15q21.1 --> 15q21.2). To our knowledge this is the first patient reported whose phenotype is due to partial trisomy 9q, and complex interstitial deletions of 15q, not involving the Prader-Willi/Angelman region and encompassing the critical region 15q21q25. We provide correlation between the clinical findings of our patient and the phenotype of the 9q34 duplication syndrome, the 15q21, and the 15q25 deletion syndromes.

Preimplantation genetic diagnosis for chromosomal rearrangements with the use of array comparative genomic hybridization at the blastocyst stage.

OBJECTIVE: To establish the value of array comparative genomic hybridization (CGH) for preimplantation genetic diagnosis (PGD) in embryos of translocation carriers in combination with vitrification and frozen embryo transfer in nonstimulated cycles. DESIGN: Retrospective data analysis study. SETTING: Academic centers for reproductive medicine and genetics. PATIENT(S): Thirty-four couples undergoing PGD for chromosomal rearrangements from October 2013 to December 2015. INTERVENTION(S): Trophectoderm biopsy at day 5 or day 6 of embryo development and subsequently whole genome amplification and array CGH were performed. MAIN OUTCOME MEASURE(S): This approach revealed a high occurrence of aneuploidies and structural rearrangements unrelated to the parental rearrangement. Nevertheless, we observed a benefit in pregnancy rates of these couples. RESULT(S): We detected chromosomal abnormalities in 133/207 embryos (64.2% of successfully amplified), and 74 showed a normal microarray profile (35.7%). In 48 of the 133 abnormal embryos (36.1%), an unbalanced rearrangement originating from the parental translocation was identified. Interestingly, 34.6% of the abnormal embryos (46/133) harbored chromosome rearrangements that were not directly linked to the parental translocation in question. We also detected a combination of unbalanced parental-derived rearrangements and aneuploidies in 27 of the 133 abnormal embryos (20.3%). CONCLUSION(S): The use of trophectoderm biopsy at the blastocyst stage is less detrimental to the survival of the embryo and leads to a more reliable estimate of the genomic content of the embryo than cleavage-stage biopsy. In this small cohort PGD study, we describe the successful implementation of array CGH analysis of blastocysts in patients with a chromosomal rearrangement to identify euploid embryos for transfer.

Comparative evaluation of despeckle filtering in ultrasound imaging of the carotid artery.

It is well-known that speckle is a multiplicative noise that degrades the visual evaluation in ultrasound imaging. The recent advancements in ultrasound instrumentation and portable ultrasound devices necessitate the need of more robust despeckling techniques for enhanced ultrasound medical imaging for both routine clinical practice and teleconsultation. The objective of this work was to carry out a comparative evaluation of despeckle filtering based on texture analysis, image quality evaluation metrics, and visual evaluation by medical experts in the assessment of 440 (220 asymptomatic and 220 symptomatic) ultrasound images of the carotid artery bifurcation. In this paper a total of 10 despeckle filters were evaluated based on local statistics, median filtering, pixel homogeneity, geometric filtering, homomorphic filtering, anisotropic diffusion, nonlinear coherence diffusion, and wavelet filtering. The results of this study suggest that the first order statistics filter lsmv, gave the best performance, followed by the geometric filter gf4d, and the homogeneous mask area filter lsminsc. These filters improved the class separation between the asymptomatic and the symptomatic classes based on the statistics of the extracted texture features, gave only a marginal improvement in the classification success rate, and improved the visual assessment carried out by the two experts. More specifically, filters lsmv or gf4d can be used for despeckling asymptomatic images in which the expert is interested mainly in the plaque composition and texture analysis; and filters lsmv, gf4d, or lsminsc can be used for the despeckling of symptomatic images in which the expert is interested in identifying the degree of stenosis and the plaque borders. The proper selection of a despeckle filter is very important in the enhancement of ultrasonic imaging of the carotid artery. Further work is needed to evaluate at a larger scale and in clinical practice the performance of the proposed despeckle filters in the automated segmentation, texture analysis, and classification of carotid ultrasound imaging.

Ultrasound imaging in the analysis of carotid plaque morphology for the assessment of stroke.

The aim of this chapter is to summarise the recent advances in ultrasonic plaque characterisation and to evaluate the efficacy of computer aided diagnosis based on neural and statistical classifiers using as input texture and morphological features. Several classifiers like the K-Nearest Neighbour (KNN) the Probabilistic Neural Network (PNN) and the Support Vecton Machine (SVM) are evaluated resulting to a diagnostic accuracy up to 71.2%.

Shallow whole genome sequencing is well suited for the detection of chromosomal aberrations in human blastocysts.

OBJECTIVE: To add evidence that massive parallel sequencing (MPS) is a valuable substitute for array comparative genomic hybridization (arrayCGH) with a resolution that is more appropriate for preimplantation genetic diagnosis (PGD) in translocation carriers. DESIGN: Study of diagnostic accuracy. SETTING: University hospital. PATIENT(S): Fifteen patients with a balanced structural rearrangement were included in the study: eight reciprocal translocations, four Robertsonian translocations, two inversions, and one insertional translocation. INTERVENTION(S): Trophectoderm biopsy was performed on 47 blastocysts. MAIN OUTCOME MEASURE(S): In the current study, shallow whole genome MPS on a NextSeq500 (Illumina) and Ion Proton (Life Technologies) instrument was performed in parallel on 47 whole genome amplified trophectoderm samples. Data analyses were performed using the QDNAseq algorithm implemented in Vivar. RESULT(S): In total, 5 normal and 42 abnormal embryos were analyzed. All aberrations previously detected with arrayCGH could be readily detected in the MPS data using both technologies and were correctly identified. The smallest detected abnormality was a ∼ 4.5 Mb deletion/duplication. CONCLUSION(S): This study demonstrates that shallow whole genome sequencing can be applied efficiently for the detection of numerical and structural chromosomal aberrations in embryos, equaling or even exceeding the resolution of the routinely used microarrays.

Whole genome amplification with SurePlex results in better copy number alteration detection using sequencing data compared to the MALBAC method.

Current whole genome amplification (WGA) methods lead to amplification bias resulting in over- and under-represented regions in the genome. Nevertheless, certain WGA methods, such as SurePlex and subsequent arrayCGH analysis, make it possible to detect copy number alterations (CNAs) at a 10 Mb resolution. A more uniform WGA combined with massive parallel sequencing (MPS), however, could allow detection at higher resolution and lower cost. Recently, MALBAC, a new WGA method, claims unparalleled performance. Here, we compared the well-established SurePlex and MALBAC WGA for their ability to detect CNAs in MPS generated data and, in addition, compared PCR-free MPS library preparation with the standard enrichment PCR library preparation. Results showed that SurePlex amplification led to more uniformity across the genome, allowing for a better CNA detection with less false positives compared to MALBAC amplified samples. An even more uniform coverage was observed in samples following a PCR-free library preparation. In general, the combination of SurePlex and MPS led to the same chromosomal profile compared to a reference arrayCGH from unamplified genomic DNA, underlining the large potential of MPS techniques in CNA detection from a limited number of DNA material.

Mutation-free baby born from a mitochondrial encephalopathy, lactic acidosis and stroke-like syndrome carrier after blastocyst trophectoderm preimplantation genetic diagnosis.

To investigate the applicability of preimplantation genetic diagnosis (PGD), we used trophectoderm (TE) biopsy to determine the mutation load in a 35-year-old female with mitochondrial encephalopathy, lactic acidosis and stroke-like syndrome (MELAS). Transfer of a mutation-free blastocyst gave birth to a healthy boy with undetectable mutation in any of the analyzed tissues. We found strong correlation among TE cells (r=0.90) within blastocysts and also between cytoplasmic fragments and TE (r=0.95). This is the first case of mutation-free baby born from a MELAS patient after TE biopsy and supports the applicability of blastocyst PGD for patients with mtDNA disorders to establish healthy offspring.

A review of noninvasive ultrasound image processing methods in the analysis of carotid plaque morphology for the assessment of stroke risk.

Noninvasive ultrasound imaging of carotid plaques allows for the development of plaque-image analysis methods associated with the risk of stroke. This paper presents several plaque-image analysis methods that have been developed over the past years. The paper begins with a review of clinical methods for visual classification that have led to standardized methods for image acquisition, describes methods for image segmentation and denoising, and provides an overview of the several texture-feature extraction and classification methods that have been applied. We provide a summary of emerging trends in 3-D imaging methods and plaque-motion analysis. Finally, we provide a discussion of the emerging trends and future directions in our concluding remarks.

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

BACKGROUND: The 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. RESULTS: Three 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%). CONCLUSIONS: The 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.

Cryptic genomic imbalances in patients with de novo or familial apparently balanced translocations and abnormal phenotype.

BACKGROUND: Carriers of apparently balanced translocations are usually phenotypically normal; however in about 6% of de novo cases, an abnormal phenotype is present. In the current study we investigated 12 patients, six de novo and six familial, with apparently balanced translocations and mental retardation and/or congenital malformations by applying 1 Mb resolution array-CGH. In all de novo cases, only the patient was a carrier of the translocation and had abnormal phenotype. In five out of the six familial cases, the phenotype of the patient was abnormal, although the karyotype appeared identical to other phenotypically normal carriers of the family. In the sixth familial case, all carriers of the translocations had an abnormal phenotype. RESULTS: Chromosomal and FISH analyses suggested that the rearrangements were "truly balanced" in all patients. However, array-CGH, revealed cryptic imbalances in three cases (3/12, 25%), two de novo (2/12, 33.3%) and one familial (1/12, 16.6%). The nature and type of abnormalities differed among the cases. In the first case, what was identified as a de novo t(9;15)(q31;q26.1), a complex rearrangement was revealed involving a ~6.1 Mb duplication on the long arm of chromosome 9, an ~10 Mb deletion and an inversion both on the long arm of chromosome 15. These imbalances were located near the translocation breakpoints. In the second case of a de novo t(4;9)(q25;q21.2), an ~6.6 Mb deletion was identified on the short arm of chromosome 7 which is unrelated to the translocation. In the third case, of a familial, t(4;7)(q13.3;p15.3), two deletions of ~4.3 Mb and ~2.3 Mb were found, each at one of the two translocation breakpoints. In the remaining cases the translocations appeared balanced at 1 Mb resolution. CONCLUSION: This study investigated both de novo and familial apparently balanced translocations unlike other relatively large studies which are mainly focused on de novo cases. This study provides additional evidence that cryptic genomic imbalances are common in patients with abnormal phenotype and "apparently balanced" translocations not only in de novo but can also occur in familial cases. The use of microarrays with higher resolution such as oligo-arrays may reveal that the frequency of cryptic genomic imbalances among these patients is higher.