Integrated Genotyping Strategies for Uncovering Detailed Haplotype Structures and Characterization of DMD Duplications.

A manifesting female carrier of Duchenne muscular dystrophy: Importance of genetics for the dystrophinopathies.

Study question To evaluate diagnosis value of optical genome mapping (OGM) for potential preimplantation genetic testing in chromosomal structural rearrangement (PGT-SR) patients with cryptic chromosomal rearrangement. Summary answer OGM is an efficient method for cryptic chromosomal rearrangement detection. However, SVs near telomere and centromere regions could hardly be reported with this method. What is known already About 5∼10% infertility or sterility people carry balanced chromosomal structural variations (SVs) with high risks of miscarriage and birth defects resulting from imbalanced gametes when producing fetus. However, some balanced cryptic SVs with specific location or small fragments cannot be detected by traditional cytogenetic techniques like G-banding testing or CNV-seq. Currently, several optimized techniques are able to detect typically cryptic structural abnormality. OGM, the molecular genetic detection technique relying on new principle in recent years, have overcome the shortcomings of former techniques and are potentially capable to detect both routine and cryptic SVs well. Study design, size, duration From 2019 to 2022, 12 couples in The First Affiliated Hospital of Sun Yat-sen University and The First People’s Hospital of Yunnan Province with history of offspring birth defect, recurrent unexplained recurrent miscarriage or unexpected copy number variation in embryos from previous PGT cycles were included for diagnosis or re-analysis. Participants/materials, setting, methods Peripheral blood lymphocytes from these patients were collected and detected by OGM or traditional karyotype analysis(G-400 banding), FISH, CNV-seq, third generation sequencing(Nanopore). The results of traditional genetic test and OGM were compared to evaluate the accuracy of OGM detection of chromosomal structure vatiation. Main results and the role of chance In the 11 couples bearing adverse pregnancy or childbirth defect with normal karyotype by G-banding, OGM discovered novel chromosome translocations or inversion in 9 couples (81.81%). In 1 couple with t(4:17)(q12;p13) diagnosed by G-banding, OGM detect an additional chromosomal translocation of t(17;19)(q12;p13), which produced the repeated CNVs in chromosome19 observed in embryos from two previous PGT cycles. For small fragment translocation, terminal translocation and inversion, OGM is able to detected balanced structure abnormal of 500kbs. The OGM diagnosis were confirmed by FISH or third generation sequencing (Nanopore) or embryo mutation. The breaking position reported by OGM and Nanopore were comparable. In sum, OGM reported further information of balanced SVs in 83.33% (10/12) potential patients with duplication and depletion in embryos or abortus. Limitations, reasons for caution Due to the limitation of principle, the structural abnormalities of some special locations (centromere, secondary constriction, etc.) cannot be detected. OGM has high sensitivity and many mutations, which requires examiners to have strong ability to analyze results and genetic counseling. The price of OGM also limits the promotion and application. Wider implications of the findings OGM can provide more precious result for SVs, and PGT-SR can definitely improve the outcome of certain population with cryptic chromosomal rearrangements. The shortcoming of OGM has not been overcame yet, so it is suggested as auxiliary method beside conventional G-binding in certain circumstances in ART population. Trial registration number Not applicable

Genetic counseling in direct-to-consumer exome sequencing: a case report.

Unveiling Clinical Potential: Exploring Cytogenomic Aberrations through Optical Genomic Mapping in Multiple Myeloma

Application of Next-Generation Cytogenetics in a Clinical Laboratory for Diagnostic Work-up of Myelodysplastic Syndromes/Neoplasms (MDS)

Chromosomal structural variations (SVs) play an important role in the formation of human cancers, including leukemias. However, many complex SVs cannot be identified by conventional tools, including karyotyping, fluorescence in situ hybridization, microarrays, and multiplex ligation-dependent probe amplification (MLPA). Optical genome mapping (OGM) and whole genome sequencing (WGS) were employed to analyze five leukemia samples with SVs detected by karyotyping, MLPA, and RNA sequencing (RNA-seq). OGM was performed using the Saphyr chip on a Bionano Saphyr system. Copy number variation and rare variant assembly analyses were performed with Bionano software v3.7. WGS was analyzed by the Manta program for SVs. The leukemia samples had an average of 477 insertions, 457 deletions, and 32 inversions, which were significantly greater than those of the normal blood samples (p = 0.016, 0.028, and 0.028, respectively). In Case 1, OGM detected a sequential translocation between chromosomes 5, 8, 12, and 21 and ETV6::RUNX1 and BCAT1::BAALC gene fusions. Case 2 had two pathogenic SVs and a BCR::ABL1 fusion. Case 3 had one pathogenic SV and an IGH::DUSP22 fusion. Case 4 had two pathogenic SVs and a CBFB::MYH11 fusion. Case 5 had an STIL::TAL1 fusion. All breakpoint sequences were defined by WGS. An IGH::DUX4 fusion previously found by RNA-seq in Case 3 was not confirmed because DUX4, which has multiple pseudogenes, was refractory to OGM and WGS analyses. OGM is a fundamental tool that complements G-banding analysis in identifying complex SVs in leukemia samples, and WGS effectively closes the gaps in OGM mapping.

Whole genome sequencing (WGS) improves Mendelian disorder diagnosis over whole exome sequencing (WES); however, additional diagnostic yields and costs remain undefined. We investigated differences between diagnostic and cost outcomes of WGS and WES in a cohort with suspected Mendelian disorders. WGS was performed in 38 WES-negative families derived from a 64 family Mendelian cohort that previously underwent WES. For new WGS diagnoses, contemporary WES reanalysis determined whether variants were diagnosable by original WES or unique to WGS. Diagnostic rates were estimated for WES and WGS to simulate outcomes if both had been applied to the 64 families. Diagnostic costs were calculated for various genomic testing scenarios. WGS diagnosed 34% (13/38) of WES-negative families. However, contemporary WES reanalysis on average 2 years later would have diagnosed 18% (7/38 families) resulting in a WGS-specific diagnostic yield of 19% (6/31 remaining families). In WES-negative families, the incremental cost per additional diagnosis using WGS following WES reanalysis was AU$36,710 (£19,407;US$23,727) and WGS alone was AU$41,916 (£22,159;US$27,093) compared to WES-reanalysis. When we simulated the use of WGS alone as an initial genomic test, the incremental cost for each additional diagnosis was AU$29,708 (£15,705;US$19,201) whereas contemporary WES followed by WGS was AU$36,710 (£19,407;US$23,727) compared to contemporary WES. Our findings confirm that WGS is the optimal genomic test choice for maximal diagnosis in Mendelian disorders. However, accepting a small reduction in diagnostic yield, WES with subsequent reanalysis confers the lowest costs. Whether WES or WGS is utilised will depend on clinical scenario and local resourcing and availability.

Tandem duplication and triplication in BRCA1: revisiting the large genomic rearrangements via optical genome mapping.

Optical Genome Mapping Reclassifies Patients with Intermediate Risk Acute Myeloid Leukemia

The use of fetal exome sequencing in prenatal diagnosis: a points to consider document of the American College of Medical Genetics and Genomics (ACMG)

Background and ObjectiveParental chromosomal structural variations (SVs) represent a primary genetic factor contributing to recurrent spontaneous abortion (RSA). Individuals carrying SVs with complex chromosomal rearrangements (CCRs) typically exhibit a normal phenotype but are at an increased risk of miscarriage. Current standard clinical detection methods are insufficient for the identification and interpretation of all SV types, particularly complex and occult SVs, thereby presenting a significant challenge for clinical genetic counseling. Leveraging the high-resolution capabilities of optical genome mapping (OGM) technology, this study aims to rapidly and accurately identify complex SVs in RSA couples. Furthermore, it seeks to conduct an in-depth analysis of the genetic information within the breakpoint regions, thereby providing a more comprehensive scientific foundation for genetic counseling of RSA couples at both the cellular and genetic levels.Material and MethodsThis study involved the selection of nine subjects from two families who underwent genetic counseling at our hospital. Family 1 comprised a couple with the wife as a SVs carrier, and both her parents and brother were simultaneously analyzed for chromosomal karyotype. Family 2 included a couple with the husband as the SVs carrier, with his parents also undergoing chromosomal karyotype analysis. For SVs carriers whose karyotype analysis did not elucidate the recombination pattern, optical genome mapping (OGM) technology was utilized for further investigation, followed by Sanger sequencing to validate the OGM findings.ResultsIn Family 1, only the wife was identified as an SVs carrier. Initial chromosomal karyotype analysis suggested a karyotype of 46,XX,t (5; 6;8; 13; 15) (?). However, OGM analysis ultimately confirmed the karyotype as 46,XY,der (5)t (5; 13) (q35.2; q21.32), der (6)t (6; 8) (q25.3; q13.1)ins (6; 13) (q25.3; q21.32q21.33),der (8)t (6; 8) (q26; q13.1)ins (8; 13) (q13.1; q21.33q22.1),der (13)t (13; 15) (q21.32; q26.1)ins (13; 6) (q21.32; q25.3q26), der (15)t (5; 15) (q35.2; q26.1). Furthermore, OGM identified a novel translocation variant of the KIF7 gene that is associated with recurrent miscarriage. In Family 2, both the husband and his maternal parent were identified as SVs carriers. Nuclear type analysis revealed a karyotype of 46,XY,?t (1; 6) (q42; p21) (husband) and 46,XX,?t (1; 2) (p31.1; q24.1),?t (1; 6) (q42; p21) (mother). Through OGM detection and analysis, the final karyotype was determined to be 46,XY,ins (1; 6) (q42.2; p22.3p11.3) (husband) and 46,XX,der (1)t (1; 2) (p31.1; q24.1)ins (1; 6) (q42.2; p22.3p11.3), der (2) t (1; 2), der (6)ins (1; 6) (mother).ConclusionOGM technology facilitates the rapid and precise identification of complex chromosomal structural variations, effectively overcoming the limitations associated with traditional karyotype G-banding techniques in detecting intricate and cryptic SVs. This advancement substantially enhances the diagnostic rates of genetic etiology in patients experiencing RSA. The present study elucidates the specific manifestations of complex SVs using OGM technology, accurately pinpointing breakpoints and interpreting affected gene information. This provides novel reference approaches and evidence for disease assessment and genetic counseling in RSA patients. However, it is important to acknowledge certain limitations of this research: the study’s inclusion of only two RSA family cohorts (comprising nine participants) may limit the generalizability of its conclusions due to the small sample size, necessitating further validation through large-scale studies. Additionally, the causal relationship between KIF7 gene dysfunction and recurrent miscarriage remains to be experimentally verified in subsequent research.

Optical Genome Mapping Combined with High-Throughput Sequencing Is Effective for the Diagnostic and Prognostic Genomic Classification of Acute Myeloid Leukemia and Myelodysplastic Neoplasms

Background: Neuroblastoma is the most common extracranial solid tumour in children, accounting for 15% of paediatric cancer deaths. Multiple genetic abnormalities have been identified as prognostically significant in neuroblastoma patients. Optical genome mapping (OGM) is a novel cytogenetic technique used to detect structural variants, which has not previously been tested in neuroblastoma. We used OGM to identify copy number and structural variants (SVs) in neuroblastoma which may have been missed by standard cytogenetic techniques. Methods: Five neuroblastoma cell lines (SH-SY5Y, NBLW, GI-ME-N, NB1691 and SK-N-BE2(C)) and two neuroblastoma tumours were analysed using OGM with the Bionano Saphyr® instrument. The results were analysed using Bionano Access software and compared to previous genetic analyses including G-band karyotyping, FISH (fluorescent in situ hybridisation), single-nucleotide polymorphism (SNP) array and RNA fusion panels for cell lines, and SNP arrays and whole genome sequencing (WGS) for tumours. Results: OGM detected copy number abnormalities found using previous methods and provided estimates for absolute copy numbers of amplified genes. OGM identified novel SVs, including fusion genes in two cell lines of potential clinical significance. Conclusions: OGM can reliably detect clinically significant structural and copy number variations in a single test. OGM may prove to be more time- and cost-effective than current standard cytogenetic techniques for neuroblastoma.

Rapid advances in DNA sequencing technologies have made it increasingly cost-effective to obtain accurate and timely large-scale genomic sequence data on individuals (short read massively parallel or next generation [next-gen]). A next-gen molecular diagnostic approach that has seen rapid deployment in the clinic over the last year is exome sequencing. Whole exome sequencing covers all protein-coding genes in the genome (≈1.1% of genome), and an exome test for a single patient generates ≈6 gigabases (109 bp) of DNA sequence data. A key challenge facing routine use of next-gen data in patient diagnosis and management is data interpretation. What sequence variant findings are relevant to diagnosis (pathogenic mutations)? What sequence variant findings are relevant to clinical care but not necessarily to patient diagnosis (clinically actionable incidental data)? What sequence information should be stored, and where can it be stored? This review provides a tutorial on current approaches to answering these questions. A recent landmark study showed that application of next-gen sequencing to a large cohort of idiopathic dilated cardiomyopathy patients found ≈27% of patients to show mutations of the titin gene, the most complex gene in the genome (363 exons). We use titin in cardiomyopathy as an exemplar for explaining next-gen sequencing approaches and data interpretation. Decreasing sequencing costs and broad dissemination of next-generation (next-gen) equipment and expertise are increasing availability of massively parallel sequencing of patient DNA samples (short read massively parallel or next-gen sequencing).1,2 Most rapidly expanding is exome sequencing, where all protein-coding sequences (exons) are selected from total genomic DNA and selectively sequenced.3 Alternative approaches to next-gen sequencing include targeted sequencing (TS) and whole genome (complete genome) sequencing. Currently, marketed targeted Sanger sequencing panels using traditional individual exon-by-exon sequencing remain expensive and time consuming, and massively parallel next-gen approaches are beginning to supplant …

Becker Muscular Dystrophy (BMD) was first described in 1955 by Peter Emil Becker,1 and has been the subject of recent interest2 including a study by Janneke van den Bergen and...