Detection Of Chromosomal Aberrations Research Articles

Optical Genome Mapping for Applications in Repeat Expansion Disorders.

Short tandem repeat (STR) expansions are associated with more than 60 genetic disorders. The size and stability of these expansions correlate with the severity and age of onset of the disease. Therefore, being able to accurately detect the absolute length of STRs is important. Current diagnostic assays include laborious lab experiments, including repeat-primed PCR and Southern blotting, that still cannot precisely determine the exact length of very long repeat expansions. Optical genome mapping (OGM) is a cost-effective and easy-to-use alternative to traditional cytogenetic techniques and allows the comprehensive detection of chromosomal aberrations and structural variants >500 bp in length, including insertions, deletions, duplications, inversions, translocations, and copy number variants. Here, we provide methodological guidance for preparing samples and performing OGM as well as running the analysis pipelines and using the specific repeat expansion workflows to determine the exact repeat length of repeat expansions expanded beyond 500 bp. Together these protocols provide all details needed to analyze the length and stability of any repeat expansion with an expected repeat size difference from the expected wild-type allele of >500 bp. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Genomic ultra-high-molecular-weight DNA isolation, labeling, and staining Basic Protocol 2: Data generation and genome mapping using the Bionano Saphyr® System Basic Protocol 3: Manual De Novo Assembly workflow Basic Protocol 4: Local guided assembly workflow Basic Protocol 5: EnFocus Fragile X workflow Basic Protocol 6: Molecule distance script workflow.

Диагностический потенциал регуляторных не кодирующих белок РНК при хроническом лимфоцитарном лейкозе

This paper reviews current knowledge about regulatory non-coding protein RNAs (ncRNAs) involved in the pathogenesis of chronic lymphocytic leukemia (CLL) and their potential capabilities as diagnostic markers. Diversity of clinical course as well as absence of detectable chromosomal aberrations and somatic mutations in 20 % of patients increase the interest to study the epigenetic aspects of pathogenesis. In this context, ncRNAs are believed to be promising diagnostic markers since their expression is commonly tissue-specific and they are quite stable in body fluids. Among the regulatory ncRNAs involved in the CLL pathogenesis, microRNAs and long (lncRNAs) have been most studied, whereas ring-like, or circulatory, ncRNAs (circRNAs) require further analysis. Aberrant expression of ncRNAs may account for the resistance to treatment in CLL patients without detected genomic abnormalities. Bioinformatics analysis of RNA sequencing databases allows to isolate novel candidate ncRNA molecules, including those associated with RNA-mediated suppression of the Piwi protein-interacting transposons. This paper proposes new independent predictive models based on the expression of 2 (LNC-KIA1755-4, LNC-IRF2-32-LNCRNA), 4 (miR-125b, miR-15b, miR-181c, miR-412), and 6 (PRKCQ, TRG.AS1, LNC00467, LNC01096, PCAT6, SBF2.AS1) simultaneously assessed different ncRNAs. Since risk- and stage classification of hematological malignancies is performed not only on the basis of clinical but also molecular genetic markers, the monitoring of regulatory ncRNA expression can provide an additional tool for more effective stratification of patients. The present review is concerned with the methodology issues in analytical procedures which impede widespread use of laboratory ncRNA tests.

M-FISH as a sensitive tool for detecting chromosome aberrations induced by low levels of radiation

M-FISH as a sensitive tool for detecting chromosome aberrations induced by low levels of radiation

Detection of chromosome aberrations in 17 054 individuals with fertility problems and their subsequent assisted reproductive technology treatments in Central China.

How do the types and frequency of chromosome aberrations in couples in central China affect fertility and ART treatment? Men with chromosome aberrations or polymorphisms have an increased risk of semen quality impairment and infertility, and couples affected by reciprocal translocations had a lower pregnancy rate compared with other chromosome aberrations. Karyotyping is crucial for patients affected by infertility as chromosome aberrations play an important role in the etiology of male infertility. However, the influence of chromosome aberrations and polymorphisms on sperm motility and morphology remains controversial. Data on ART treatment outcomes in infertile couples affected by chromosome aberrations are insufficient. We conducted a retrospective study involving 17 054 patients affected by infertility who underwent karyotyping in our center between January 2020 and May 2022. Karyotyping was performed on 17 054 patients with reproductive failure. All patients were from the central regions of China. The following data were collected from a medical records system using patient identification numbers: couples' ages, history of pregnancy and childbirth, type of infertility, years of infertility, cause of infertility, chromosome karyotypes, semen analysis results, assisted reproductive techniques performed, and treatment outcomes of ART. The incidence of chromosome aberrations was 2.04%; 2.49% in men and 1.57% in women. By analyzing the relationships between chromosome aberrations/polymorphisms and abnormal semen parameters, we found that there were significantly higher rates of asthenozoospermia, oligospermia, and teratozoospermia among men with Robertsonian translocations and sex chromosomal structural aberrations compared with those with normal karyotypes. Higher rates of asthenozoospermia and teratozoospermia were also observed among men with autosomal reciprocal translocations. The incidence of chromosome aberrations in azoospermic men (13.75%), and in men with cryptozoospermia or severe oligospermia (6.97%) was significantly higher than that in men with mild oligospermia or normospermia (0.88-2.12%). In addition, we found that the progressive movement of sperm is impaired in men with Chromosome 21 polymorphisms compared with men with normal karyotypes (39.46% ± 20.51% vs 48.61% ± 18.76%, P = 0.026). The percentage of morphologically normal forms was lower in the chromosomal polymorphism group than in the normal karyotype group (5.01% ± 2.41% vs 5.59% ± 2.14%, P = 0.001), especially in men with polymorphisms on Chromosome 9 (enlarged Chromosome 9 heterochromatin [9qh+]: 4.48% ± 2.22% vs 5.59% ± 2.14%, P = 0.006; pericentric inversion of Chromosome 9 [inv(9)]: 5.09% ± 3.11% vs 5.59% ± 2.14%, P = 0.008). ART treatment was successful in 36.00% of couples affected by chromosome aberrations. However, couples affected by reciprocal translocations achieved a lower pregnancy rate (24.07%), which may be due to the lower euploidy rates (27.31%) when compared with that in other chromosome aberrations. First, although the initial cohort was large, chromosome aberrations were identified in a small number of patients. Second, the observational nature of the study design is limiting. Third, the couples affected by infertility in this study were all outpatients that did not undergo identical comprehensive examinations except for karyotyping, leading to the incomplete collection of medical records. Also, the population included in this study mainly focused on couples affected by infertility, which may not be included in the European Association of Urology (EAU) recommendation on male infertility. Men with chromosome aberrations or polymorphisms have an increased risk of semen quality impairment and infertility. Constitutional chromosome analysis is recommended for men affected by infertility and severe oligospermia or azoospermia to facilitate early and appropriate guidance for the most suitable treatment. Carriers of chromosome aberrations can achieve acceptable pregnancy outcomes through IVF. However, couples affected by reciprocal translocations have lower pregnancy rates, and more treatment cycles are needed before a successful pregnancy. A possible explanation may be the fewer euploid embryos obtained. This work was supported by Grant 2021YFC2700603 from the National Key Research & Development Program of China. The authors declare no conflicts of interest. N/A.

Optical genome mapping for detection of chromosomal aberrations in prenatal diagnosis.

Chromosomal aberrations are the most important etiological factors for birth defects. Optical genome mapping is a novel cytogenetic tool for detecting a broad range of chromosomal aberrations in a single assay, but relevant clinical feasibility studies of optical genome mapping in prenatal diagnosis are limited. We retrospectively performed optical genome mapping analysis of amniotic fluid samples from 34 fetuses with various clinical indications and chromosomal aberrations detected through standard-of-care technologies, including karyotyping, fluorescence in situ hybridization, and/or chromosomal microarray analysis. In total, we analyzed 46 chromosomal aberrations from 34 amniotic fluid samples, including 5 aneuploidies, 10 large copy number variations, 27 microdeletions/microduplications, 2 translocations, 1 isochromosome, and 1 region of homozygosity. Overall, 45 chromosomal aberrations could be confirmed by our customized analysis strategy. Optical genome mapping reached 97.8% concordant clinical diagnosis with standard-of-care methods for all chromosomal aberrations in a blinded fashion. Compared with the widely used chromosomal microarray analysis, optical genome mapping additionally determined the relative orientation and position of repetitive segments for seven cases with duplications or triplications. The additional information provided by optical genome mapping will be conducive to characterizing complex chromosomal rearrangements and allowing us to propose mechanisms to explain rearrangements and predict the genetic recurrence risk. Our study highlights that optical genome mapping can provide comprehensive and accurate information on chromosomal aberrations in a single test, suggesting that optical genome mapping has the potential to become a promising cytogenetic tool for prenatal diagnosis.

False positivity in break apart fluorescence in-situ hybridization due to polyploidy.

In-situ hybridization (ISH) is a diagnostic tool in the detection of chromosomal anomalies, which has important implications for diagnosis, classification and prediction of cancer therapy in various diseases. Certain thresholds of number of cells showing an aberrant pattern are commonly used to declare a sample as positive for genomic rearrangements. The phenomenon of polyploidy can be misleading in the interpretation of break apart fluorescence in-situ hybridization (FISH). The aim of this study is to investigate the impact of cell size and ploidy on FISH results. In sections of varying thickness of control liver tissue and non-small cell lung cancer cases, nuclear size was measured and the number of MET chromogenic ISH and ALK FISH (liver) or ALK and ROS1 FISH (lung cancer) signals was manually counted and quantified. In liver cell nuclei the number of FISH/chromogenic ISH signals increases with nuclear size related to physiological polyploidy and is related to section thickness. In non-small cell lung cancer cases tumour cells with higher ploidy levels and nuclear size have an increased chance of single signals. Furthermore, additional lung cancer samples with borderline ALK FISH results were examined with a commercial kit for rearrangements. No rearrangements could be demonstrated, proving a false positive ALK FISH result. In case of polyploidy there is an increased likelihood of false positivity when using break apart FISH probes. Therefore, we state that prescribing one single cut-off in FISH is inappropriate. In polyploidy, the currently proposed cut-off should only be used with caution and the result should be confirmed by an additional technique.

Severity Scoring Cutoff for MLPA and Its Diagnostic Yield in 332 North Indian Children with Developmental Delay.

Chromosomal aberrations/rearrangements are the most common cause of intellectual disability (ID), developmental delay (DD), and congenital malformations. Traditionally, karyotyping has been the investigation of choice in such cases, with the advantage of being cheap and easily accessible, but with the caveat of the inability to detect copy number variations of sizes less than 5 Mb. Chromosomal microarray can solve this problem, but again the problems of expense and poor availability are major challenges in developing countries. The purpose of this study is to find the utility of multiplex ligation-dependent probe amplification (MLPA) as a middle ground, in a resource-limited setting. We also attempted to establish an optimum cutoff for the de Vries score, to enable physicians to decide between these tests on a case-to-case basis, using only clinical data. A total of 332 children with DD/ID with or without facial dysmorphism and congenital malformations were studied by MLPA probe sets P245. Assessment of clinical variables concerning birth history, facial dysmorphism, congenital malformations, and family history was done. We also scored the de Vries scoring for all the patients to find a suitable cutoff for MLPA screening. In our study, the overall detection rate of MLPA was 13.5% (45/332). The majority of patients were DiGeorge's syndrome with probe deletion in 22q11.21 in 3.3% (11/332) followed by 15q11.2 del in 3.6% (12/332, split between Angelman's and Prader-Willi's syndromes). Also, 3.0% (10/332) of patients were positive for Williams-Beuren's syndrome 7q11.23, 1.8% (6/332) for Wolf--Hirschhorn's syndrome 4p16.3, 1.2% (4/332) for 1p36 deletion, and 1% for each trichorhinophalangeal syndrome type I 8q23.3 duplication syndrome and cri du chat syndrome. The optimum cutoff of de Vries score for MLPA testing in children with ID and/or dysmorphism came out to be 2.5 (rounded off to 3) with a sensitivity of 82.2% and specificity of 66.7%. This is the largest study from India for the detection of chromosomal aberrations using MLPA common microdeletion kit P245. Our study suggests that de Vries score with a cutoff of 3 or more can be used to offer MLPA as the first tier test for patients with unexplained ID, with or without facial dysmorphism and congenital malformations.

Single Nucleotide Polymorphism Array Versus Karyotype for Prenatal Diagnosis in Fetuses With Abnormal Ultrasound: A Systematic Review and Meta-analysis

(Abstracted from Eur J Obstet Gynecol Reprod Biol 2022;271:235–244) Chromosomal aberrations occur at a frequency of approximately 1 in 150 to 200 live births and are one of the most important causes of congenital anomalies and miscarriages. The introduction of chromosomal microarray (CMA) in the 1990s advanced the detection of chromosomal aberrations to include small copy number variants that previously were not detectable when only karyotype was used.

Classical and Molecular Cytogenetics: The Principle and Application of Involved Techniques

Cytogenetics is the study of chromosomes. The involved techniques have an indispensable role in chromosome aberration detection. Taking the invention of in situ hybridization (ISH) as a dividing line, all techniques are categorized into classical and molecular cytogenetics. Typical techniques in classical cytogenetics are chromosome banding and flow karyotyping. Branches of ISH, such as fluorescence in situ hybridization, spectral karyotyping, and comparative genomic hybridization, are often classified as molecular cytogenetics. All of these techniques have provided researchers with either quantitative or qualitative data to aid in the analysis of chromosomes and have made significant contributions to the fields of prenatal detection, prognostic, diagnostics, oncology, etc.

FISH with Whole Chromosome Painting Probes.

Some types of chromosome aberrations are not easily identified by the traditional Giemsa staining. It usually needs some banding technique and skilled person's eye. Whole chromosome painting FISHprobe can stain designated entire chromosomes or domains in metaphase chromosomes or interphase nuclei, respectively. It allows to visually identify translocations, deletions, or amplifications of specific chromosomes. Once chromosomes are stained, even non-skilled researchers can easily identify those chromosome aberrations. Whole chromosome painting FISH has higher sensitivity to detect chromosome aberrations. This chapter introduces the method for whole chromosome painting FISH staining.

The role of individual blood flow parameters through ductus venosus in the first and second trimesters of pregnancy in predicting the condition of the fetus and newborn.

To predict fetal and neonatal outcome during pregnancy based on detailed analysis of ductus venosus blood flow velocities in first and second-trimester fetuses. A retrospective analysis was made in 680 patients with single pregnancies in years 2015 and 2016. The following ductus venosus blood flow velocities in first and second-trimester were analyzed: S-wave velocity, D-wave velocity, a-wave velocity, Tmax velocity, PIV. Results were divided into sub-groups with reduced value, normal value and increased value and compared with fetal and neonatal condition. The relationship between the increased PIV value in the first trimester of pregnancy and an increased risk of chromosomal aberrations was observed, whereas the increased DV PI value in the second trimester of pregnancy with reduced A -wave were associated with a higher incidence of FGR. No correlation between the remaining DV blood flow velocities in the first and second trimester of pregnancy and the more frequent occurrence of fetal and neonatal complications has been confirmed. The increased DV PIV is a good prognostic tool for the detection of chromosomal aberrations in first trimester of pregnancy. In the second trimester, the increased DV PIV and the reduced A- wave velocity correlate with the fetal growth restriction. Ductus venosus seems to be an indirect indicator of intrauterine hypoxia with moderate prognostic value for adverse obstetric outcomes.

Molecular and cytogenetic characterization of myelodysplastic syndromes in cell-free DNA

AbstractMolecular and cytogenetic studies are essential for diagnosis and prognosis in patients with myelodysplastic syndromes (MDSs). Cell-free DNA (cfDNA) analysis has been reported to be a reliable noninvasive approach for detecting molecular abnormalities in MDS; however, there is limited information about cytogenetic alterations and monitoring in cfDNA. We assessed the molecular and cytogenetic profile of a cohort of 70 patients with MDS by next-generation sequencing (NGS) of cfDNA and compared the results to sequencing of paired bone marrow (BM) DNA. Sequencing of BM DNA and cfDNA showed a comparable mutational profile (92.1% concordance), and variant allele frequencies (VAFs) strongly correlated between both sample types. Of note, SF3B1 mutations were detected with significantly higher VAFs in cfDNA than in BM DNA. NGS and microarrays were highly concordant in detecting chromosomal alterations although with lower sensitivity than karyotype and fluorescence in situ hybridization. Nevertheless, all cytogenetic aberrations detected by NGS in BM DNA were also detected in cfDNA. In addition, we monitored molecular and cytogenetic alterations and observed an excellent correlation between the VAFs of mutations in BM DNA and cfDNA across multiple matched time points. A decrease in the cfDNA VAFs was detected in patients responding to therapy, but not in nonresponding patients. Of note, cfDNA analysis also showed cytogenetic evolution in 2 nonresponsive cases. In summary, although further studies with larger cohorts are needed, our results support the analysis of cfDNA as a promising strategy for performing molecular characterization, detection of chromosomal aberrations and monitoring of patients with MDS.

Plant Cytogenetics in the Micronuclei Investigation-The Past, Current Status, and Perspectives.

Cytogenetic approaches play an essential role as a quick evaluation of the first genetic effects after mutagenic treatment. Although labor-intensive and time-consuming, they are essential for the analyses of cytotoxic and genotoxic effects in mutagenesis and environmental monitoring. Over the years, conventional cytogenetic analyses were a part of routine laboratory testing in plant genotoxicity. Among the methods that are used to study genotoxicity in plants, the micronucleus test particularly represents a significant force. Currently, cytogenetic techniques go beyond the simple detection of chromosome aberrations. The intensive development of molecular biology and the significantly improved microscopic visualization and evaluation methods constituted significant support to traditional cytogenetics. Over the past years, distinct approaches have allowed an understanding the mechanisms of formation, structure, and genetic activity of the micronuclei. Although there are many studies on this topic in humans and animals, knowledge in plants is significantly limited. This article provides a comprehensive overview of the current knowledge on micronuclei characteristics in plants. We pay particular attention to how the recent contemporary achievements have influenced the understanding of micronuclei in plant cells. Together with the current progress, we present the latest applications of the micronucleus test in mutagenesis and assess the state of the environment.

Value of Nuchal Translucency in Detection of Chromosomal Aberration in Vietnam Population

Objective: To examine the sensitivity and specificity of different thresholds of nuchal translucency in diagnosis of chromosomal defects. Study Design: This is a longitudinal study of pregnant women have first trimester screening and ultrasound in center of diagnostic antenatal of national hospital of obstetrics and gynecology. A follow-up was made to identify, in all singleton pregnancies in both group of which fetal karyotyping was made and group of normal fetuses. The threshold for nuchal translucency was divided in to above the 95th percentile, the 99th percentile, the 3.0mm and 2.5 MoM of nuchal translucency. The sensitivity and specificity ware calculated in order to diagnosis the chromosomal abbreviation. Results: The research identified 2645 fetuses, 743 amniocentesis (28%). There is 32.4% fetus has NT ≥ the 95th percentile, 28.6% ≥ 2.5mm percentile, 22.3% ≥ 3.0mm, 16.6% above 2.5 MoM. The fetal karyotype was abnormal in 157 (5.8%) pregnancies. The popular conditions were found including trisomy 21(52.2%). Then structural rearrangements occupied 31.2%. Other chromosomes like 13,18,21 occupied 12.7%. The abnormal of sex chromosome was smallest proportion with only 3.8%. At the 95th percentile of nuchal translucency has the highest sensitivity in detection of chromosomal defects (99.4%) but the threshold 2.5mm has a better detection rate (20.4%). The cut off 3.0mm has a better positive prediction rate (22.3%) but could detect less defects (only 132/157 abbreviation). The threshold 2.5xMoM had the highest specificity (86.4%) but lowest sensitivity (only 65%). Conclusion: In fetuses with increased nuchal translucency, more than a half of the chromosomally abnormal group is affected by defects other than trisomy 21 (52.2%). Using threshold 2.5mm helps detect more 23 chromosomal defects in comparison with the threshold 3.0mm and it had the highest average of sensitivity and specificity (87.25%).

Residual risk for clinically significant copy number variants in low-risk pregnancies, following exclusion of noninvasive prenatal screening–detectable findings

Chromosomal microarray analysis detects a clinically significant amount of copy number variants in approximately 1% of low-risk pregnancies. As the constantly growing use of noninvasive prenatal screening has facilitated the detection of chromosomal aberrations, defining the rate of abnormal chromosomal microarray analysis findings following normal noninvasive prenatal screening is of importance for making informed decisions regarding prenatal testing and screening options. To calculate the residual risk for clinically significant copy number variants following theoretically normal noninvasive prenatal screening. The chromosomal microarray results of all pregnancies undergoing amniocentesis between the years 2013 and 2021 in a large hospital-based laboratory were collected. Pregnancies with sonographic anomalies, abnormal maternal serum screening, or multiple fetuses were excluded. Clinically significant (pathogenic and likely pathogenic) copy number variants were divided into the following: 3-noninvasive prenatal screening-detectable (trisomies 13, 18, and 21), 5- noninvasive prenatal screening-detectable (including sex chromosome aberrations), 5-noninvasive prenatal screening and common microdeletion-detectable (including 1p36.3-1p36.2, 4p16.3-4p16.2, 5p15.3-5p15.1, 15q11.2-15q13.1, and 22q11.2 deletions), and genome-wide noninvasive prenatal screening-detectable (including variants >7 Mb). The theoretical residual risk for clinically significant copy number variants was calculated following the exclusion of noninvasive prenatal screening-detectable findings. Of the 7235 pregnancies, clinically significant copy number variants were demonstrated in 87 cases (1.2%). The residual risk following theoretically normal noninvasive prenatal screening was 1.07% (1/94) for 3-noninvasive prenatal screening, 0.78% (1/129) for 5- noninvasive prenatal screening, 0.74% (1/136) for 5- noninvasive prenatal screening including common microdeletions, and 0.68% (1/147) for genome-wide noninvasive prenatal screening. In the subgroup of 4048 pregnancies with advanced maternal age, the residual risk for clinically significant copy number variants following theoretically normal noninvasive prenatal screening ranged from 1.36% (1/73) for 3- noninvasive prenatal screening to 0.82% (1/122) for genome-wide noninvasive prenatal screening. In 3187 pregnancies of women <35 years, this residual risk ranged from 0.69% (1/145) for 3- noninvasive prenatal screening to 0.5% (1/199) for genome-wide noninvasive prenatal screening. The residual risk of clinically significant copy number variants in pregnancies without structural sonographic anomalies is appreciable and depends on the noninvasive prenatal screening extent and maternal age. This knowledge is important for the patients, obstetricians, and genetic counselors to facilitate informed decisions regarding prenatal testing and screening options.

Optical genome mapping enables constitutional chromosomal aberration detection

Chromosomal aberrations including structural variations (SVs) are a major cause of human genetic diseases. Their detection in clinical routine still relies on standard cytogenetics. Drawbacks of these tests are a very low resolution (karyotyping) and the inability to detect balanced SVs or indicate the genomic localization and orientation of duplicated segments or insertions (copy number variant [CNV] microarrays). Here, we investigated the ability of optical genome mapping (OGM) to detect known constitutional chromosomal aberrations. Ultra-high-molecular-weight DNA was isolated from 85 blood or cultured cells and processed via OGM. A de novo genome assembly was performed followed by structural variant and CNV calling and annotation, and results were compared to known aberrations from standard-of-care tests (karyotype, FISH, and/or CNV microarray). In total, we analyzed 99 chromosomal aberrations, including seven aneuploidies, 19 deletions, 20 duplications, 34 translocations, six inversions, two insertions, six isochromosomes, one ring chromosome, and four complex rearrangements. Several of these variants encompass complex regions of the human genome involved in repeat-mediated microdeletion/microduplication syndromes. High-resolution OGM reached 100% concordance compared to standard assays for all aberrations with non-centromeric breakpoints. This proof-of-principle study demonstrates the ability of OGM to detect nearly all types of chromosomal aberrations. We also suggest suited filtering strategies to prioritize clinically relevant aberrations and discuss future improvements. These results highlight the potential for OGM to provide a cost-effective and easy-to-use alternative that would allow comprehensive detection of chromosomal aberrations and structural variants, which could give rise to an era of "next-generation cytogenetics."

How genomics is changing the practice of prenatal testing.

New genomic laboratory technology namely microarrays and high throughput sequencing (HTS) as well as a steady progress in sonographic image capture and processing have changed the practice of prenatal diagnosis during the last decade fundamentally. Pregnancies at high risk for common trisomies are reliably identified by non-invasive prenatal testing (NIPT) and expert sonography has greatly improved the assessment of the fetal phenotype. Preconceptional comprehensive carrier screening using HTS is available for all parents, if they should wish to do so. A definite fetal diagnosis, however, will still require invasive testing for most conditions. Chromosomal microarrays (CMA) have greatly enhanced the resolution in the detection of chromosome anomalies and other causal copy number variations (CNV). Gene panel or whole exome sequencing (WES) is becoming the routine follow up of many anomalies detected by ultrasound after CNVs have been excluded. The benefits and limitations of the various screening as well as diagnostic options are perceived as complex by many who find it challenging to cope with the need for immediate choices. The communication of facts to ensure an informed decision making is obviously a growing challenge with the advent of the new genomic testing options. This contribution provides an overview of the current practice and policies in Switzerland.

Rare chromosomal aberrations detected in children with multiple congenital anomalies: utility of multiple ligation dependant probe amplification for developing countries

Chromosomal aberrations are an important cause of multiple malformation syndromes. Multiple ligation-dependent probe amplification (MLPA) a molecular cytogenetic technique has been suggested as a screening tool for the detection of chromosomal aberrations in resource-limited settings. MLPA can detect chromosomal microdeletions or duplications at approximately 40 chromosomal regions in a single experiment. Several MLPA kits are available to target the chromosomal regions of interest. In the present study, we aimed to detect the yield and utility of MLPA in a cohort of children with multiple malformations and developmental delay. MLPA was performed using kits P245, P070 and P036. The overall yield of MLPA in our cohort was 8%. The manuscript describes very rare and interesting cases of congenital anomalies, such as severe buphthalmos and biphalangeal fingers with a chromosomal etiology. The study demonstrates the usefulness of MLPA as screening technique for chromosomal aberrations in children with multiple malformation syndromes, especially for developing countries such as India.

28. Comparison of different SNP microarray designs and whole genome sequencing for cytogenetic testing

Since the landmark consensus statement paper published in 20101, microarray testing has become the primary tool for detection of chromosomal aberrations of various sizes. In fact, high-density SNP arrays have now become one of the most popular forms of cytogenetic testing due to their ability to detect regions of homozygosity (which is clinically useful) in addition to detection of copy number variants (CNVs). We have performed extensive analytical analysis of the designs from 10 SNP arrays offered by Thermo Fisher/Affymetrix and Illumina.

Artificial Intelligence Substantially Supports Chromosome Banding Analysis Maintaining Its Strengths in Hematologic Diagnostics Even in the Era of Newer Technologies

Background: Chromosome banding analysis (CBA) is one of the most important techniques in diagnostics and prognostication in hematologic neoplasms. CBA is still a challenging method with very labor-intensive wet lab processes and karyotyping that requires highly skilled and experienced specialists for tumor cytogenetics. Short turnaround times (TAT) are becoming increasingly important to enable genetics-based treatment stratification at diagnosis. Aim: Improve TAT and quality of CBA by automated wet lab processes and AI-based algorithms for automatic karyotyping. Methods: In the last 15 years the CBA workflow has gradually been automated with focus on the wet lab and metaphase capturing processes. Now, a retrospective unselected digital data set of 100,000 manually arranged karyograms (KG) with normal karyotype (NKG) from routine diagnostics was used to train a deep neural network (DNN) classifier to automatically determine the class/number and orientation of the respective chromosomes (AI based classifier normal, AI-CN). With a total of 6 Mio parameters, the DNN uses two distinct output layers to simultaneously predict the chromosome number (24 classes) and the angle that is required to rotate the chromosome in its correct, vertical position (360 classes). Training of the DNN took 16 days on a Nvidia RTX 2080 Ti graphic card with 4352 cores. AI-CN was implemented into the routine workflow (including ISO 15189) after 7 months of development and intensive testing. Results: The AI-CN was tested by highly experienced staff in an independent prospective validation set of 500 NKG: 22,675/23,000 chromosomes (98.6%) were correctly assigned by AI-CN. In 369/500 (73.8%) of cells all chromosomes were correctly assigned, in an additional 20% only 2 chromosomes were interchanged. The chromosomes accounting for the majority of misclassifications were chromosomes 14 and 15 as well as 4 and 5, which are difficult to distinguish in poor quality metaphases also for humans. The 1st AI-CN was implemented into routine diagnostics in August 2019 and the 2nd AI-CN - optimized for chromosome orientation - was used since November 2019. Since then more than 17,500 cases have been processed with AI-CN (&amp;gt;350,000 metaphases) in routine diagnostics resulting in the following benefits: 1) Reduced working time: an experienced cytogeneticist needs - depending on chromosome quality - between 1 and 3 minutes to arrange a KG, while AI-CN needs only 1 second and the cytogeneticist about 30 seconds to review the KG. 2) Shorter TAT: The proportion of cases reported within 5 days increased from 30% before AI-CN (2019) to 36% with AI-CN1 (2019) and 45% with AI-CN2 (2019/2020), while the proportion of cases reported &amp;gt;7 days was reduced to 28%, 21%, and 17%, respectively (figure). Using AI-CN for aberrant karyotypes results in correct assignment of normal chromosomes and thus also correct KG in cases with solely numerical chromosome abnormalities. Derivative chromosomes derived from structural abnormalities (SA) that differ clearly from any normal chromosome are not automatically assigned but are left out for manual classification. Thus, even in cases with SA, using AI-CN saves time. To allow AI based SA assignment, two additional classifiers normal/aberrant (CNA) were built: AI-CNA1 was trained on 54,634 KG encompassing 10 different SA (AKG) and 100,000 NKG and AI-CNA2 was trained on all AKG and an equal number of NKG. First validation tests are promising and optimization is ongoing. Once the CNA has been optimized, a standardized high quality of chromosome aberration detection is feasible. A fully automated separation of chromosomes is currently in progress and will reduce the TAT by another 12-24 hours. In a fully automated workflow the detection of small subclones can be further optimized by increasing today's standard of 20 metaphases to several hundred, even without any delay in TAT and need for additional personnel. Conclusions: Implementation of AI in CBA substantially improves the quality of results and shortens turnaround times even in comparison to highly trained and experienced cytogeneticists. In the majority of cases a complete karyotype analysis can be guaranteed within 3 to 7 days, allowing CBA based treatment strategies at diagnosis. This fully automated workflow can be implemented worldwide, is rapidly scalable, can be performed cloud based and requires in the near future fewer experienced tumor cytogeneticists. Figure Disclosures Hänselmann: MetaSystems: Current Employment. Lörch:MetaSystems: Current equity holder in private company.