Development and performance of a comprehensive targeted sequencing assay for pan-ethnic screening of carrier status.

Identifying individuals as carriers of severe disease traits enables informed decision making about reproductive options. Although carrier screening has traditionally been based on ethnicity, the increasing ethnic admixture in the general population argues for the need for pan-ethnic carrier screening assays. Highly multiplexed mutation panels allow for rapid and efficient testing of hundreds of mutations concurrently. We report the development of the Pan-Ethnic Carrier Screening assay, a targeted sequencing assay for routine screening that simultaneously detects 461 common mutations in 91 different genes underlying severe, early-onset monogenic disorders. Mutation selection was aided by the use of an extensive mutation database from a clinical laboratory with expertise in newborn screening and lysosomal storage disease testing. The assay is based on the Affymetrix GeneChip microarray platform but generates genomic DNA sequence as the output. Analytical sensitivity and specificity, using genomic DNA from archived control cultures and from clinical specimens, was found to be >99% for all mutation types. This targeted sequencing assay has advantages over multiplex PCR and next-generation sequencing assays, including accuracy of mutation detection over a range of mutation types and ease of analysis and reporting of results.

Molecular diagnostic testing for congenital disorders of glycosylation (CDG): detection rate for single gene testing and next generation sequencing panel testing.

Congenital disorders of glycosylation (CDG) are comprised of over 60 disorders with the majority of defects residing within the N-glycosylation pathway. Approximately 20% of patients do not survive beyond five years of age due to widespread organ dysfunction. A diagnosis of CDG is based on abnormal glycosylation of transferrin but this method cannot identify the specific gene defect. For many individuals diagnosed with CDG the gene defect remains unknown. To improve the molecular diagnosis of CDG we developed molecular testing for 25 CDG genes including single gene testing and next generation sequencing (NGS) panel testing. From March 2010 through November 2012, a total of 94 samples were referred for single gene testing and 68 samples were referred for NGS panel testing. Disease causing mutations were identified in 24 patients resulting in a molecular diagnosis rate of 14.8%. Coverage of the 24 CDG genes using panel testing and whole exome sequencing (WES) was compared and it was determined that many exons of these genes were not adequately covered using a WES approach and a panel approach may be the preferred first option for CDG patients. A collaborative effort between physicians, researchers and diagnostic laboratories will be very important as NGS testing using panels and exome becomes more widespread. This technology will ultimately improve the molecular diagnosis of patients with CDG in hard to solve cases.

Comprehensive mutation analysis for congenital muscular dystrophy: a clinical PCR-based enrichment and next-generation sequencing panel.

The congenital muscular dystrophies (CMDs) comprise a heterogeneous group of heritable muscle disorders with often difficult to interpret muscle pathology, making them challenging to diagnose. Serial Sanger sequencing of suspected CMD genes, while the current molecular diagnostic method of choice, can be slow and expensive. A comprehensive panel test for simultaneous screening of mutations in all known CMD-associated genes would be a more effective diagnostic strategy. Thus, the CMDs are a model disorder group for development and validation of next-generation sequencing (NGS) strategies for diagnostic and clinical care applications. Using a highly multiplexed PCR-based target enrichment method (RainDance) in conjunction with NGS, we performed mutation detection in all CMD genes of 26 samples and compared the results with Sanger sequencing. The RainDance NGS panel showed great consistency in coverage depth, on-target efficiency, versatility of mutation detection, and genotype concordance with Sanger sequencing, demonstrating the test's appropriateness for clinical use. Compared to single tests, a higher diagnostic yield was observed by panel implementation. The panel's limitation is the amplification failure of select gene-specific exons which require Sanger sequencing for test completion. Successful validation and application of the CMD NGS panel to improve the diagnostic yield in a clinical laboratory was shown.

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.

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.

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.

A forward genetic screen in Drosophila implicates insulin signaling in age-related locomotor impairment.

Age-related locomotor impairment (ARLI) is one of the most detrimental changes that occurs during aging. Elderly individuals with ARLI are at increased risks for falls, depression and a number of other co-morbidities. Despite its clinical significance, little is known about the genes that influence ARLI. We consequently performed a forward genetic screen to identify Drosophila strains with delayed ARLI using negative geotaxis as an index of locomotor function. One of the delayed ARLI strains recovered from the screen had a P-element insertion that decreased expression of the insulin signaling gene phosphoinositide-dependent kinase 1 (PDK1) Precise excision of the P-element insertion reverted PDK1 expression and ARLI to the same as control flies, indicating that disruption of PDK1 leads to delayed ARLI. Follow-up studies showed that additional loss of function mutations in PDK1 as well as loss of function alleles of two other insulin signaling genes, Dp110 and Akt (the genes for the catalytic subunit of phosphoinositide 3-kinase and AKT), also forestalled ARLI. Interestingly, only some of the strains with delayed ARLI had elevated resistance to paraquat, indicating that enhanced resistance to this oxidative stressor is not required for preservation of locomotor function across age. Our studies implicate insulin signaling as a key regulator of ARLI in Drosophila.

Sod2 knockdown in the musculature has whole-organism consequences in Drosophila.

Oxidative damage to cell macromolecules by reactive oxygen species is associated with numerous diseases and aging. In Drosophila, RNAi-mediated silencing of the mitochondrial antioxidant manganese superoxide dismutase (SOD2) throughout the body dramatically reduces life span, accelerates senescence of locomotor function, and enhances sensitivity to applied oxidative stress. Here, we show that Sod2 knockdown in the musculature alone is sufficient to cause the shortened life span and accelerated locomotor declines observed with knockdown of Sod2 throughout the body, indicating that Sod2 deficiency in muscle is central to these phenotypes. Knockdown of Sod2 in the muscle also increased caspase activity (a marker for apoptosis) and caused a mitochondrial pathology characterized by swollen mitochondria, decreased mitochondrial content, and reduced ATP levels. These findings indicate that Sod2 plays a crucial role in the musculature in Drosophila and that the consequences of SOD2 loss in this tissue extend to the viability of the organism as a whole.

Genetic and environmental factors impact age-related impairment of negative geotaxis in Drosophila by altering age-dependent climbing speed.

Age-related locomotor impairment in humans is important clinically because it is associated with several co-morbidities and increased risk of death. One of the hallmarks of age-related locomotor impairment in humans is a decrease in walking speed with age. Genetically tractable model organisms such as Drosophila are essential for delineating mechanisms underlying age-related locomotor impairment and age-related decreases in locomotor speed. Negative geotaxis, the ability of flies to move vertically when startled, is a common measure of locomotor behavior that declines with age in Drosophila. Toward further developing Drosophila as a model for age-related locomotor impairment, we investigated whether negative geotaxis reflects climbing or a combination of climbing and other behaviors such as flying and jumping. Additionally, we investigated whether locomotor speed in negative geotaxis assays declines with age in flies as found for walking speed in humans. We find that the vast majority of flies climb during negative geotaxis assays and that removal of hind legs, but not wings, impairs the behavior. We also find that climbing speed decreases with age in four wild type genetic backgrounds, in flies housed at different temperatures, and in control and long-lived flies harboring a mutation in OR83b. The decreases in climbing speed correlate with the age-related impairments in the distance climbed. These studies establish negative geotaxis in Drosophila as a climbing behavior that declines with age due to a decrease in climbing speed. Age-related decreases in locomotor speed are common attributes of locomotor senescence in flies and humans.