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.

Assessment and Clinical Utility of a Non-Next-Generation Sequencing-Based Non-Invasive Prenatal Testing Technology.

Background: Rolling-circle replication (RCR) is a novel technology that has not been applied to cell-free DNA (cfDNA) testing until recently. Given the cost and simplicity advantages of this technology compared to other platforms currently used in cfDNA analysis, an assessment of RCR in clinical laboratories was performed. Here, we present the first validation study from clinical laboratories utilizing RCR technology. Methods: 831 samples from spontaneously pregnant women carrying a singleton fetus, and 25 synthetic samples, were analyzed for the fetal risk of trisomy 21 (T21), trisomy 18 (T18) and trisomy 13 (T13), by three laboratories on three continents. All the screen-positive pregnancies were provided post-test genetic counseling and confirmatory diagnostic invasive testing (e.g., amniocentesis). The screen-negative pregnancies were routinely evaluated at birth for fetal aneuploidies, using newborn examinations, and any suspected aneuploidies would have been offered diagnostic testing or confirmed with karyotyping. Results: The study found rolling-circle replication to be a highly viable technology for the clinical assessment of fetal aneuploidies, with 100% sensitivity for T21 (95% CI: 82.35-100.00%); 100.00% sensitivity for T18 (71.51-100.00%); and 100.00% sensitivity for T13 analyses (66.37-100.00%). The specificities were >99% for each trisomy (99.7% (99.01-99.97%) for T21; 99.5% (98.62-99.85%) for T18; 99.7% (99.03-99.97%) for T13), along with a first-pass no-call rate of 0.93%. Conclusions: The study showed that using a rolling-circle replication-based cfDNA system for the evaluation of the common aneuploidies would provide greater accuracy and clinical utility compared to conventional biochemical screening, and it would provide comparable results to other reported cfDNA methodologies.

DDOST mutations identified by whole-exome sequencing are implicated in congenital disorders of glycosylation.

Congenital disorders of glycosylation (CDG) are inherited autosomal-recessive diseases that impair N-glycosylation. Approximately 20% of patients do not survive beyond the age of 5 years old as a result of widespread organ dysfunction. Although most patients receive a CDG diagnosis based on abnormal glycosylation of transferrin, this test cannot provide a genetic diagnosis; indeed, many patients with abnormal transferrin do not have mutations in any known CDG genes. Here, we combined biochemical analysis with whole-exome sequencing (WES) to identify the genetic defect in an untyped CDG patient, and we found a 22 bp deletion and a missense mutation in DDOST, whose product is a component of the oligosaccharyltransferase complex that transfers the glycan chain from a lipid carrier to nascent proteins in the endoplasmic reticulum lumen. Biochemical analysis with three biomarkers revealed that N-glycosylation was decreased in the patient's fibroblasts. Complementation with wild-type-DDOST cDNA in patient fibroblasts restored glycosylation, indicating that the mutations were pathological. Our results highlight the power of combining WES and biochemical studies, including a glyco-complementation system, for identifying and confirming the defective gene in an untyped CDG patient. This approach will be very useful for uncovering other types of CDG as well.

Assessment of clinical analytical sensitivity and specificity of next-generation sequencing for detection of simple and complex mutations.

BACKGROUND: Detecting mutations in disease genes by full gene sequence analysis is common in clinical diagnostic laboratories. Sanger dideoxy terminator sequencing allows for rapid development and implementation of sequencing assays in the clinical laboratory, but it has limited throughput, and due to cost constraints, only allows analysis of one or at most a few genes in a patient. Next-generation sequencing (NGS), on the other hand, has evolved rapidly, although to date it has mainly been used for large-scale genome sequencing projects and is beginning to be used in the clinical diagnostic testing. One advantage of NGS is that many genes can be analyzed easily at the same time, allowing for mutation detection when there are many possible causative genes for a specific phenotype. In addition, regions of a gene typically not tested for mutations, like deep intronic and promoter mutations, can also be detected. RESULTS: Here we use 20 previously characterized Sanger-sequenced positive controls in disease-causing genes to demonstrate the utility of NGS in a clinical setting using standard PCR based amplification to assess the analytical sensitivity and specificity of the technology for detecting all previously characterized changes (mutations and benign SNPs). The positive controls chosen for validation range from simple substitution mutations to complex deletion and insertion mutations occurring in autosomal dominant and recessive disorders. The NGS data was 100% concordant with the Sanger sequencing data identifying all 119 previously identified changes in the 20 samples. CONCLUSIONS: We have demonstrated that NGS technology is ready to be deployed in clinical laboratories. However, NGS and associated technologies are evolving, and clinical laboratories will need to invest significantly in staff and infrastructure to build the necessary foundation for success.

Detection limit of intragenic deletions with targeted array comparative genomic hybridization.

BACKGROUND: Pathogenic mutations range from single nucleotide changes to deletions or duplications that encompass a single exon to several genes. The use of gene-centric high-density array comparative genomic hybridization (aCGH) has revolutionized the detection of intragenic copy number variations. We implemented an exon-centric design of high-resolution aCGH to detect single- and multi-exon deletions and duplications in a large set of genes using the OGT 60 K and 180 K arrays. Here we describe the molecular characterization and breakpoint mapping of deletions at the smaller end of the detectable range in several genes using aCGH. RESULTS: The method initially implemented to detect single to multiple exon deletions, was able to detect deletions much smaller than anticipated. The selected deletions we describe vary in size, ranging from over 2 kb to as small as 12 base pairs. The smallest of these deletions are only detectable after careful manual review during data analysis. Suspected deletions smaller than the detection size for which the method was optimized, were rigorously followed up and confirmed with PCR-based investigations to uncover the true detection size limit of intragenic deletions with this technology. False-positive deletion calls often demonstrated single nucleotide changes or an insertion causing lower hybridization of probes demonstrating the sensitivity of aCGH. CONCLUSIONS: With optimizing aCGH design and careful review process, aCGH can uncover intragenic deletions as small as dozen bases. These data provide insight that will help optimize probe coverage in array design and illustrate the true assay sensitivity. Mapping of the breakpoints confirms smaller deletions and contributes to the understanding of the mechanism behind these events. Our knowledge of the mutation spectra of several genes can be expected to change as previously unrecognized intragenic deletions are uncovered.

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.

DMD-Associated Dilated Cardiomyopathy: Genotypes, Phenotypes, and Phenocopies.

BACKGROUND: Variants in the DMD gene, that encodes the cytoskeletal protein, dystrophin, cause a severe form of dilated cardiomyopathy (DCM) associated with high rates of heart failure, heart transplantation, and ventricular arrhythmias. Improved early detection of individuals at risk is needed. METHODS: Genetic testing of 40 male probands with a potential X-linked genetic cause of primary DCM was undertaken using multi-gene panel sequencing, multiplex polymerase chain reaction, and array comparative genomic hybridization. Variant location was assessed with respect to dystrophin isoform patterns and exon usage. Telomere length was evaluated as a marker of myocardial dysfunction in left ventricular tissue and blood. RESULTS: Four pathogenic/likely pathogenic DMD variants were found in 5 probands (5/40: 12.5%). Only one rare variant was identified by gene panel testing with 3 additional multi-exon deletion/duplications found following targeted assays for structural variants. All of the pathogenic/likely pathogenic DMD variants involved dystrophin exons that had percent spliced-in scores >90, indicating high levels of constitutive expression in the human adult heart. Fifteen DMD variant-negative probands (15/40: 37.5%) had variants in autosomal genes including TTN, BAG3, LMNA, and RBM20. Myocardial telomere length was reduced in patients with DCM irrespective of genotype. No differences in blood telomere length were observed between genotype-positive family members with/without DCM and controls. CONCLUSIONS: Primary genetic testing using multi-gene panels has a low yield and specific assays for structural variants are required if DMD-associated cardiomyopathy is suspected. Distinguishing X-linked causes of DCM from autosomal genes that show sex differences in clinical presentation is crucial for informed family management.

Targeted polymerase chain reaction-based enrichment and next generation sequencing for diagnostic testing of congenital disorders of glycosylation.

PURPOSE: Congenital disorders of glycosylation are a heterogeneous group of disorders caused by deficient glycosylation, primarily affecting the N-linked pathway. It is estimated that more than 40% of congenital disorders of glycosylation patients lack a confirmatory molecular diagnosis. The purpose of this study was to improve molecular diagnosis for congenital disorders of glycosylation by developing and validating a next generation sequencing panel for comprehensive mutation detection in 24 genes known to cause congenital disorders of glycosylation. METHODS: Next generation sequencing validation was performed on 12 positive control congenital disorders of glycosylation patients. These samples were blinded as to the disease-causing mutations. Both RainDance and Fluidigm platforms were used for sequence enrichment and targeted amplification. The SOLiD platform was used for sequencing the amplified products. Bioinformatic analysis was performed using NextGENe® software. RESULTS: The disease-causing mutations were identified by next generation sequencing for all 12 positive controls. Additional variants were also detected in three controls that are known or predicted to impair gene function and may contribute to the clinical phenotype. CONCLUSIONS: We conclude that development of next generation sequencing panels in the diagnostic laboratory where multiple genes are implicated in a disorder is more cost-effective and will result in improved and faster patient diagnosis compared with a gene-by-gene approach. Recommendations are also provided for data analysis from the next generation sequencing-derived data in the clinical laboratory, which will be important for the widespread use of this technology.

Targeted comparative genomic hybridization array for the detection of single- and multiexon gene deletions and duplications.

PURPOSE: To develop a high resolution microarray based method to detect single- and multiexons gene deletions and duplications. METHODS: We have developed a high-resolution comparative genomic hybridization array to detect single- and multiexon deletions and duplications in a large set of genes on a single microarray, using the NimbleGen 385K array with an exon-centric design. RESULTS: We have successfully developed, validated, and implemented a targeted gene comparative genomic hybridization arrays for detecting single- and multiexon deletions and duplication in autosomal and X-linked disease-associated genes. CONCLUSION: The comparative genomic hybridization arrays can be adopted readily by clinical molecular diagnostic laboratories as a rapid, cost-effective, highly sensitive, and accurate approach for the detection of single- and multiexon deletions or duplications, particularly in cases where direct sequencing fails to identify a mutation.

Microarray-based mutation detection in the dystrophin gene.

Duchenne and Becker muscular dystrophies (DMD and BMD) are X-linked recessive neuromuscular disorders caused by mutations in the dystrophin gene affecting approximately 1 in 3,500 males. The human dystrophin gene spans>2,200 kb, or roughly 0.1% of the genome, and is composed of 79 exons. The mutational spectrum of disease-causing alleles, including exonic copy number variations (CNVs), is complex. Deletions account for approximately 65% of DMD mutations and 85% of BMD mutations. Duplications occur in approximately 6 to 10% of males with either DMD or BMD. The remaining 30 to 35% of mutations consist of small deletions, insertions, point mutations, or splicing mutations, most of which introduce a premature stop codon. Laboratory analysis of dystrophin can be used to confirm a clinical diagnosis of DMD, characterize the type of dystrophin mutation, and perform prenatal testing and carrier testing for females. Current dystrophin diagnostic assays involve a variety of methodologies, including multiplex PCR, Southern blot analysis, multiplex ligation-dependent probe amplification (MLPA), detection of virtually all mutations-SSCP (DOVAM-S), and single condition amplification/internal primer sequencing (SCAIP); however, these methods are time-consuming, laborious, and do not accurately detect duplication mutations in the dystrophin gene. Furthermore, carrier testing in females is often difficult when a related affected male is unavailable. Here we describe the development, design, validation, and implementation of a high-resolution comparative genomic hybridization (CGH) microarray-based approach capable of accurately detecting both deletions and duplications in the dystrophin gene. This assay can be readily adopted by clinical molecular testing laboratories and represents a rapid, cost-effective approach for screening a large gene, such as dystrophin.

Array CGH improves detection of mutations in the GALC gene associated with Krabbe disease.

BACKGROUND: Krabbe disease is an autosomal recessive lysosomal storage disorder caused by mutations in the GALC gene. The most common mutation in the Caucasian population is a 30-kb deletion of exons 11 through 17. There are few other reports of intragenic GALC deletions or duplications, due in part to difficulties detecting them. METHODS AND RESULTS: We used gene-targeted array comparative genomic hybridization (CGH) to analyze the GALC gene in individuals with Krabbe disease in whom sequence analysis with 30-kb deletion analysis identified only one mutation. In our sample of 33 cases, traditional approaches failed to identify two pathogenic mutations in five (15.2%) individuals with confirmed Krabbe disease. The addition of array CGH deletion/duplication analysis to the genetic testing strategy led to the identification of a second pathogenic mutation in three (9.1%) of these five individuals. In all three cases, the deletion or duplication identified through array CGH was a novel GALC mutation, including the only reported duplication in the GALC gene, which would have been missed by traditional testing methodologies. We report these three cases in detail. The second mutation remains unknown in the remaining two individuals (6.1%), despite our full battery of testing. CONCLUSIONS: Analysis of the GALC gene using array CGH deletion/duplication testing increased the two-mutation detection rate from 84.8% to 93.9% in affected individuals. Better mutation detection rates are important for improving molecular diagnosis of Krabbe disease, as well as for providing prenatal and carrier testing in family members.

Assessment of target enrichment platforms using massively parallel sequencing for the mutation detection for congenital muscular dystrophy.

Sequencing individual genes by Sanger sequencing is a time-consuming and costly approach to resolve clinically heterogeneous genetic disorders. Panel testing offers the ability to efficiently and cost-effectively screen all of the genes for a particular genetic disorder. We assessed the analytical sensitivity and specificity of two different enrichment technologies, solution-based hybridization and microdroplet-based PCR target enrichment, in conjunction with next-generation sequencing (NGS), to identify mutations in 321 exons representing 12 different genes involved with congenital muscular dystrophies. Congenital muscular dystrophies present diagnostic challenges due to phenotypic variability, lack of standard access to and inherent difficulties with muscle immunohistochemical stains, and a general lack of clinician awareness. NGS results were analyzed across several parameters, including sequencing metrics and genotype concordance with Sanger sequencing. Genotyping data showed that both enrichment technologies produced suitable calls for use in clinical laboratories. However, microdroplet-based PCR target enrichment is more appropriate for a clinical laboratory, due to excellent sequence specificity and uniformity, reproducibility, high coverage of the target exons, and the ability to distinguish the active gene versus known pseudogenes. Regardless of the method, exons with highly repetitive and high GC regions are not well enriched and require Sanger sequencing for completeness. Our study demonstrates the successful application of targeted sequencing in conjunction with NGS to screen for mutations in hundreds of exons in a genetically heterogeneous human disorder.

A simple method to confirm and size deletion, duplication, and insertion mutations detected by sequence analysis.

Characterizing heterozygous insertions or deletions in genes by PCR and Sanger sequencing can be a challenge due to overlapping sequencing traces produced by overlapping templates. This is particularly problematic for clinical diagnostic laboratories, because mutations must be precisely characterized. Although the mutation detection software used by clinical diagnostic laboratories reliably identifies small insertions and deletions, overlapping deletions and insertions on opposite chromosomes, complex rearrangements, and insertions or deletions close to the primer sites may be missed. Here we describe a rapid, simple method to confirm and precisely characterize deletions and insertions using a capillary-based gel electrophoresis system. This technique has been applied to a series of patients with deletion, duplication, or insertion mutations identified by sequencing, as well as to patients with repeat tract polymorphisms, to demonstrate the utility of this method.