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We previously reported the benefit of lomustine addition to conventional chemotherapy in older acute myeloid leukemias with nonadverse chromosomal aberrations in the LAM-SA 2007 randomized clinical trial (NCT00590837). A molecular analysis of 52 genes performed in 330 patients included in this trial, 163 patients being treated with lomustine in combination with idarubicin and cytarabine and 167 without lomustine, identified 1088 mutations with an average of 3.3 mutations per patient. NPM1 , FLT3 , and DNMT3A were the most frequently mutated genes. A putative therapeutic target was identified in 178 patients (54%). Among five molecular classifications analyzed, the ELN2017 risk classification has the stronger association with the clinical evolution. Patients not treated with lomustine have an expected survival prognosis in agreement with this classification regarding the overall and event-free survivals. In strong contrast, lomustine erased the ELN2017 classification prognosis. The benefit of lomustine in nonadverse chromosomal aberrations was restricted to patients with RUNX1, ASXL1 , TP53 , and FLT3 -ITD high / NPM1 WT mutations in contrast to the intermediate and favorable ELN2017 patients. This post-hoc analysis identified a subgroup of fit elderly AML patients with intermediate cytogenetics and molecular markers who may benefit from lomustine addition to intensive chemotherapy.

Unique molecular identifiers (UMIs) associate distinct sequences with every DNA or RNA molecule and can be counted after amplification to quantify molecules in the original sample. Using UMIs, the authors obtain a digital karyotype of an individual with Down's syndrome and quantify mRNA in Drosophila melanogaster cells. Counting individual RNA or DNA molecules is difficult because they are hard to copy quantitatively for detection. To overcome this limitation, we applied unique molecular identifiers (UMIs), which make each molecule in a population distinct, to genome-scale human karyotyping and mRNA sequencing in Drosophila melanogaster . Use of this method can improve accuracy of almost any next-generation sequencing method, including chromatin immunoprecipitation–sequencing, genome assembly, diagnostics and manufacturing-process control and monitoring.

The sperm of infertile men have higher rates of chromosomal abnormalities than those of fertile men. Miscarriage rate is also higher following testicular sperm extraction combined with intracytoplasmic sperm injection (TESE-ICSI). Sperm chromosomal abnormalities are assumed to be the cause of miscarriages. Previous testicular sperm karyotyping studies have only examined a few selected chromosomes using fluorescence in situ hybridization. The aim of this study was to provide a more detailed analysis of sperm karyotyping by analyzing all chromosomes using next-generation sequencing (NGS) in clinically usable testicular sperm. Sperm discarded after clinical use was collected for NGS. Additionally, sperm were individually collected by micromanipulation from patients with obstructive azoospermia (OA) and non-obstructive azoospermia (NOA) who underwent TESE-ICSI. For comparison, ejaculated sperm from control and balanced translocation (BT) carriers were examined. Karyotyping was performed on individual sperm cells using NGS. The number of normal and aberrant sperm was compared. Seventeen patients participated in this study: control (n = 4), BT (n = 3), OA (n = 5), and NOA (n = 5). Ten sperm samples per patient were analyzed. The total acquisition rate for single sperm karyotyping was 85% (145/170). Karyotyping of sperm from the BT group revealed sperm with unbalanced chromosomes derived from carrier translocations. Among the NOA group, 7/41 (17%) sperm samples exhibited aberrant karyotypes, whereas no aberrant sperm were identified in the control and OA groups. Individual differences were observed in the frequency of sperm chromosomal abnormalities among patients with NOA. In conclusion, sperm chromosomal abnormalities are frequently observed in patients with NOA even after sperm selection for clinical use. As the frequency of chromosomal abnormalities varies among patients with NOA, single sperm sequencing may help identify patients with NOA most likely to benefit from PGT-A.

Chromosomal microarray analysis (CMA) is currently considered first-tier testing in pediatric care and prenatal diagnosis owing to its high diagnostic sensitivity for chromosomal imbalances. The aim of this study was to determine the efficacy and diagnostic power of CMA in both fresh and formalin-fixed paraffin-embedded (FFPE) samples of products of conception (POCs). Over a 44-month period, 8,118 consecutive samples were received by our laboratory for CMA analysis. This included both fresh (76.4%) and FFPE samples (22.4%), most of which were ascertained for recurrent pregnancy loss and/or spontaneous abortion (83%). The majority of samples were evaluated by a whole-genome single-nucleotide polymorphism (SNP)-based array (81.6%); the remaining samples were evaluated by array-comparative genomic hybridization (CGH). A successful result was obtained in 7,396 of 8,118 (91.1%), with 92.4% of fresh tissue samples and 86.4% of FFPE samples successfully analyzed. Clinically significant abnormalities were identified in 53.7% of specimens (3,975 of 7,396), 94% of which were considered causative. Analysis of POC specimens by karyotyping fails in 20–40% of cases. SNP-based CMA is a robust platform, with successful results obtained in >90% of cases. SNP-based CMA can identify aneuploidy, polyploidy, whole-genome homozygosity, segmental genomic imbalances, and maternal cell contamination, thus maximizing sensitivity and decreasing false-negative results. Understanding the etiology of fetal loss enables clarification of recurrence risk and assists in determining appropriate management for future family planning.

Intellectual disability (ID) is a measurable phenotypic consequence of genetic and environmental factors. In this study, we prospectively assessed the diagnostic yield of genomic tools (molecular karyotyping, multi-gene panel and exome sequencing) in a cohort of 337 ID subjects as a first-tier test and compared it with a standard clinical evaluation performed in parallel. Standard clinical evaluation suggested a diagnosis in 16% of cases (54/337) but only 70% of these (38/54) were subsequently confirmed. On the other hand, the genomic approach revealed a likely diagnosis in 58% ( n =196). These included copy number variants in 14% ( n =54, 15% are novel), and point mutations revealed by multi-gene panel and exome sequencing in the remaining 43% (1% were found to have Fragile-X). The identified point mutations were mostly recessive ( n =117, 81%), consistent with the high consanguinity of the study cohort, but also X-linked ( n =8, 6%) and de novo dominant ( n =19, 13%). When applied directly on all cases with negative molecular karyotyping, the diagnostic yield of exome sequencing was 60% (77/129). Exome sequencing also identified likely pathogenic variants in three novel candidate genes ( DENND5A , NEMF and DNHD1 ) each of which harbored independent homozygous mutations in patients with overlapping phenotypes. In addition, exome sequencing revealed de novo and recessive variants in 32 genes ( MAMDC2, TUBAL3, CPNE6, KLHL24, USP2, PIP5K1A, UBE4A, TP53TG5, ATOH1, C16ORF90, SLC39A14, TRERF1, RGL1, CDH11, SYDE2, HIRA, FEZF2, PROCA1, PIANP, PLK2, QRFPR, AP3B2, NUDT2, UFC1, BTN3A2, TADA1, ARFGEF3 , FAM160B1 , ZMYM5 , SLC45A1 , ARHGAP33 and CAPS2 ), which we highlight as potential candidates on the basis of several lines of evidence, and one of these genes ( SLC39A14 ) was biallelically inactivated in a potentially treatable form of hypermanganesemia and neurodegeneration. Finally, likely causal variants in previously published candidate genes were identified ( ASTN1, HELZ , THOC6 , WDR45B, ADRA2B and CLIP1 ), thus supporting their involvement in ID pathogenesis. Our results expand the morbid genome of ID and support the adoption of genomics as a first-tier test for individuals with ID.

In recent years, optical genome mapping (OGM) has developed into a highly promising method of detecting large-scale structural variants in human genomes. It is capable of detecting structural variants considered difficult to detect by other current methods. Hence, it promises to be feasible as a first-line diagnostic tool, permitting insight into a new realm of previously unknown variants. However, due to its novelty, little experience with OGM is available to infer best practices for its application or to clarify which features cannot be detected. In this study, we used the Saphyr system (Bionano Genomics, San Diego, CA, USA), to explore its capabilities in human genetic diagnostics. To this end, we tested 14 DNA samples to confirm a total of 14 different structural or numerical chromosomal variants originally detected by other means, namely, deletions, duplications, inversions, trisomies, and a translocation. Overall, 12 variants could be confirmed; one deletion and one inversion could not. The prerequisites for detection of similar variants were explored by reviewing the OGM data of 54 samples analyzed in our laboratory. Limitations, some owing to the novelty of the method and some inherent to it, were described. Finally, we tested the successful application of OGM in routine diagnostics and described some of the challenges that merit consideration when utilizing OGM as a diagnostic tool.

Key messageAn Oligo-FISH barcode system was developed for two model legumes, allowing the identification of all cowpea and common bean chromosomes in a single FISH experiment, and revealing new chromosome rearrangements. The FISH barcode system emerges as an effective tool to understand the chromosome evolution of economically important legumes and their related species.Current status on plant cytogenetic and cytogenomic research has allowed the selection and design of oligo-specific probes to individually identify each chromosome of the karyotype in a target species. Here, we developed the first chromosome identification system for legumes based on oligo-FISH barcode probes. We selected conserved genomic regions between Vigna unguiculata (Vu, cowpea) and Phaseolus vulgaris (Pv, common bean) (diverged ~ 9.7–15 Mya), using cowpea as a reference, to produce a unique barcode pattern for each species. We combined our oligo-FISH barcode pattern with a set of previously developed FISH probes based on BACs and ribosomal DNA sequences. In addition, we integrated our FISH maps with genome sequence data. Based on this integrated analysis, we confirmed two translocation events (involving chromosomes 1, 5, and 8; and chromosomes 2 and 3) between both species. The application of the oligo-based probes allowed us to demonstrate the participation of chromosome 5 in the translocation complex for the first time. Additionally, we detailed a pericentric inversion on chromosome 4 and identified a new paracentric inversion on chromosome 10. We also detected centromere repositioning associated with chromosomes 2, 3, 5, 7, and 9, confirming previous results for chromosomes 2 and 3. This first barcode system for legumes can be applied for karyotyping other Phaseolinae species, especially non-model, orphan crop species lacking genomic assemblies and cytogenetic maps, expanding our understanding of the chromosome evolution and genome organization of this economically important legume group.

Purpose To evaluate the diagnostic yield of chromosomal microarray (CMA) in pregnancies with normal ultrasound. Methods This retrospective cohort analysis included all pregnancies with normal ultrasound undergoing CMA testing between the years 2010 and 2016. We calculated the rate of detection of clinically significant CMA findings in the whole cohort and according to various indications. Results Of 5541 CMA analyses, clinically significant findings were yielded in 78 cases (1.4%). Of these, 31 (39.7%) variants could have theoretically been detected by karyotyping (e.g., sized above 10 Mb), and 28 (35.9%) by noninvasive prenatal screening aimed at five common aneuploidies. Of the 47 submicroscopic findings detectable by CMA only, the majority (37 cases, 78.7%) represented known recurrent syndromes. Detection of clinically significant CMA findings in women with no indication for invasive testing was 0.76% (21/2752), which was significantly lower compared with 1.8% in advanced maternal age group (41/2336), 2.8% in abnormal biochemical serum screening (6/211), and 4.1% (10/242) in fetuses with sonographic soft markers. Conclusion Clinically significant CMA aberrations are detected in 1 of 71 pregnancies with normal ultrasound, and in 1 of 131 women with no indication for invasive testing. Thus, CMA might be recommended a first-tier test in pregnancies with normal ultrasound.

Purpose: Our aim was to compare the accuracy of family- or disease-specific targeted haplotyping and direct mutation-detection strategies with the accuracy of genome-wide mapping of the parental origin of each chromosome, or karyomapping, by single-nucleotide polymorphism genotyping of the parents, a close relative of known disease status, and the embryo cell(s) used for preimplantation genetic diagnosis of single-gene defects in a single cell or small numbers of cells biopsied from human embryos following in vitro fertilization. Methods: Genomic DNA and whole-genome amplification products from embryo samples, which were previously diagnosed by targeted haplotyping, were genotyped for single-nucleotide polymorphisms genome-wide detection and retrospectively analyzed blind by karyomapping. Results: Single-nucleotide polymorphism genotyping and karyomapping were successful in 213/218 (97.7%) samples from 44 preimplantation genetic diagnosis cycles for 25 single-gene defects with various modes of inheritance distributed widely across the genome. Karyomapping was concordant with targeted haplotyping in 208 (97.7%) samples, and the five nonconcordant samples were all in consanguineous regions with limited or inconsistent haplotyping results. Conclusion: Genome-wide karyomapping is highly accurate and facilitates analysis of the inheritance of almost any single-gene defect, or any combination of loci, at the single-cell level, greatly expanding the range of conditions for which preimplantation genetic diagnosis can be offered clinically without the need for customized test development. Genet Med 16 11, 838–845.

Preconception and prenatal genetic counseling is a well-established means of risk assessment in many parts of the world, and in recent years, an emerging concept in India. Likelihood of an offspring having autosomal recessive disorder increases based on the degree of consanguinity. Hence, genetic testing of the couple for the identification of carrier status for disease-causing variants is crucial. The purpose of this study is to understand the frequency of genetic abnormalities in consanguineous marriages by using a comprehensive genetic testing algorithm where in karyotyping, FISH, exome sequencing and microarray are used sequentially to determine the genetic etiology based on the clinical presentation and to evaluate the need and benefits of preconceptional and prenatal genetic counseling. This retrospective study includes 66 couples having consanguinity referred for genetic counseling and testing. Of the 66 couples, 58 underwent comprehensive genetic testing which included Karyotyping, Fluorescence in Situ Hybridization (FISH), Microarray and Exome sequencing based on their clinical presentation. The analyses revealed a genetic abnormality in approximately 31% and chromosomal polymorphic variations & variants of uncertain significance in 17% of the couples. Counseling in these couples helped in identifying the carrier status and enabled them to take an informed decision in subsequent pregnancies. These findings reiterate the acute need for preconception and prenatal genetic counseling services in India.