Evidence from 2100 index cases supports genome sequencing as a first-tier genetic test.

PURPOSE: Genome sequencing (GS) is one of the most comprehensive assays that interrogate single-nucleotide variants, copy number variants, mitochondrial variants, repeat expansions, and structural variants in a single assay. Despite the clear technical superiority, the full clinical utility of GS has yet to be determined. METHODS: We systematically evaluated 2100 clinical GS index cases performed in our laboratory to explore the diagnostic yield of GS as first-tier and as follow-up testing. RESULTS: The overall diagnostic yield was 28% (585/2100). The diagnostic yield for GS as the first-tier test was 26% (294/1146). Among cases with prior non-diagnostic genetic tests, GS provided a diagnosis for 27% (247/910) of cases, including 56 cases with prior exome sequencing (ES). Although re-analysis of previous ES might have resolved the diagnosis in 29 cases, diagnoses for 27 cases would have been missed because of the technical inferiority of ES. Moreover, GS further disclosed additional genetic etiology in 3 out of 44 cases with existing partial diagnosis. CONCLUSION: We present the largest-to-date GS data set of a clinically heterogeneous cohort from a single clinical laboratory. Our data demonstrate that GS should be considered as the first-tier genetic test that has the potential to shorten the diagnostic odyssey.

Testing for Facioscapulohumeral Muscular Dystrophy with Optical Genome Mapping.

The introduction of optical genome mapping has improved time constraints and a lack of specificity from previous methodologies when performing genome-wide analyses of samples. Optical genome mapping allows for the detection of structural variations, aberrations, and functionality traits from a single stained molecule of DNA. Though the preparation time is increased compared to previously utilized visualization techniques, optical genome mapping significantly reduces the time needed for analysis. Specifically, individual disease pipelines have been developed to rapidly analyze prepared samples. One of these diseases, Facioscapulohumeral Muscular Dystrophy (FSHD), is detected through quantification of the D4Z4 repeat array on chromosome 4q35. Optical genome mapping, with the ability to enumerate the repeats of the D4Z4 array, has demonstrated the capability to precisely diagnose FSHD. In this protocol, the preparation of samples and subsequent loading and analysis in an optical genome mapping system is discussed for the detection and analysis of FSHD. These methods should prove highly useful in FSHD analyses and beyond with the development of further disease analysis pipelines within the instrument. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Genomic DNA isolation, labeling, and staining Basic Protocol 2: Mapping and analysis with the Bionano Saphyr® system.

Molecular Diagnosis of Duchenne Muscular Dystrophy Using Single NGS-Based Assay.

Duchenne Muscular Dystrophy (DMD) is an X-linked inherited neuromuscular disorder caused by pathogenic variants in the dystrophin gene (DMD; locus Xp21.2). The variant spectrum of DMD is unique in that 65% of causative mutations are intragenic deletions, with intragenic duplications and point mutations (along with other sequence variants) accounting for 6% to 10% and 30% to 35%, respectively. The traditional strategy for molecular diagnostic testing for DMD involves initial screening for deletions/duplications using microarray-based comparative genomic hybridization followed by a full-sequence analysis of DMD for sequence variants. This traditional strategy is expensive and time-consuming due to the involvement of two separate tests to detect all types of variants in the DMD gene. Recent advancements in next-generation sequencing (NGS) technology and improvements in analysis algorithms related to copy number variant detection ultimately resulted in the development of a single NGS-based assay to detect all variant types, including deletions/duplications and sequence variants. This article initially discusses the strategic algorithm for establishing a molecular diagnosis of DMD and later provides detailed molecular diagnostic protocols for DMD, including an NGS-based sequencing assay with sequence and copy number variant analysis. © 2023 Wiley Periodicals LLC. Basic Protocol: Next-generation sequencing of the entire genomic sequence of the DMD gene using IDT xGen Lockdown Probes.

At-Risk Genomic Findings for Pediatric-Onset Disorders From Genome Sequencing vs Medically Actionable Gene Panel in Proactive Screening of Newborns and Children.

Importance: Although the clinical utility of genome sequencing for critically ill children is well recognized, its utility for proactive pediatric screening is not well explored. Objective: To evaluate molecular findings from screening ostensibly healthy children with genome sequencing compared with a gene panel for medically actionable pediatric conditions. Design, Setting, and Participants: This case series study was conducted among consecutive, apparently healthy children undergoing proactive genetic screening for pediatric disorders by genome sequencing (n = 562) or an exome-based panel of 268 genes (n = 606) from March 1, 2018, through July 31, 2022. Exposures: Genetic screening for pediatric-onset disorders using genome sequencing or an exome-based panel of 268 genes. Main Outcomes and Measures: Molecular findings indicative of genetic disease risk. Results: Of 562 apparently healthy children (286 girls [50.9%]; median age, 29 days [IQR, 9-117 days]) undergoing screening by genome sequencing, 46 (8.2%; 95% CI, 5.9%-10.5%) were found to be at risk for pediatric-onset disease, including 22 children (3.9%) at risk for high-penetrance disorders. Sequence analysis uncovered molecular diagnoses among 32 individuals (5.7%), while copy number variant analysis uncovered molecular diagnoses among 14 individuals (2.5%), including 4 individuals (0.7%) with chromosome scale abnormalities. Overall, there were 47 molecular diagnoses, with 1 individual receiving 2 diagnoses; of the 47 potential diagnoses, 22 (46.8%) were associated with high-penetrance conditions. Pathogenic variants in medically actionable pediatric genes were found in 6 individuals (1.1%), constituting 12.8% (6 of 47) of all diagnoses. At least 1 pharmacogenomic variant was reported for 89.0% (500 of 562) of the cohort. In contrast, of 606 children (293 girls [48.3%]; median age, 26 days [IQR, 10-67 days]) undergoing gene panel screening, only 13 (2.1%; 95% CI, 1.0%-3.3%) resulted in potential childhood-onset diagnoses, a significantly lower rate than those screened by genome sequencing (P < .001). Conclusions and Relevance: In this case series study, genome sequencing as a proactive screening approach for children, due to its unrestrictive gene content and technical advantages in comparison with an exome-based gene panel for medically actionable childhood conditions, uncovered a wide range of heterogeneous high-penetrance pediatric conditions that could guide early interventions and medical management.

Molecular Diagnosis of Facioscapulohumeral Muscular Dystrophy in Patients Clinically Suspected of FSHD Using Optical Genome Mapping.

Background and Objectives: Facioscapulohumeral muscular dystrophy (FSHD) represents the third most common muscular dystrophy in the general population and is characterized by progressive and often asymmetric muscle weakness of the face, upper extremities, arms, lower leg, and hip girdle. In FSHD type 1, contraction of the number of D4Z4 repeats to 1-10 on the chromosome 4-permissive allele (4qA) results in abnormal epigenetic derepression of the DUX4 gene in skeletal muscle. In FSHD type 2, epigenetic derepression of the DUX4 gene on the permissive allele (4qA) with normal-sized D4Z4 repeats (mostly 8-20) is caused by heterozygous pathogenic variants in chromatin modifier genes such as SMCHD1, DNMT3B, or LRIF1. We present validation of the optical genome mapping (OGM) platform for accurate mapping of the D4Z4 repeat size, followed by diagnostic testing of 547 cases with a suspected clinical diagnosis of FSHD and next-generation sequencing (NGS) of the SMCHD1 gene to identify cases with FSHD2. Methods: OGM with Bionano Genomics Saphyr and EnFocus FSHD analysis software was used to identify FSHD haplotypes and D4Z4 repeat number and compared with the gold standard of Southern blot-based diagnosis. A custom Agilent SureSelect enrichment kit was used to enrich SMCHD1, followed by NGS on an Illumina system with 100-bp paired-end reads. Copy number variants were assessed using NxClinical software. Results: We performed OGM for the diagnosis of FSHD in 547 patients suspected of FSHD between December 2019 and December 2022, including 301 male (55%) and 246 female patients (45%). Overall, 308 of the referred patients were positive for D4Z4 contraction on a permissive haplotype, resulting in a diagnosis of FSHD1. A total of 252 of 547 patients were referred for concurrent testing for FSHD1 and FSHD2. This resulted in the identification of FSHD2 in 9/252 (3.6%) patients. In our FSHD2 cohort, the 4qA allele size ranged from 8 to 18 repeats. Among FSHD1-positive cases, 2 patients had biallelic contraction and 4 patients had homozygous contraction and showed early onset of clinical features. Nine of the 308 patients (3%) positive for 4qA contraction had mosaic 4q alleles with contraction on at least one 4qA allele. The overall diagnostic yield in our cohort was 58%. Discussion: A combination of OGM to identify the FSHD haplotype and D4Z4 repeat number and NGS to identify sequence and copy number variants in the SMCHD1 gene is a practical and cost-effective option with increased precision for accurate diagnosis of FSHD types 1 and 2.