Success rates for genomic analyses of highly heterogeneous disorders can be greatly improved if a large cohort of patient data is assembled to enhance collective capabilities for accurate sequence variant annotation, analysis, and interpretation. Indeed, molecular diagnostics requires the establishment of robust data resources to enable data sharing that informs accurate understanding of genes, variants, and phenotypes. The “Mitochondrial Disease Sequence Data Resource (MSeqDR) Consortium” is a grass-roots effort facilitated by the United Mitochondrial Disease Foundation to identify and prioritize specific genomic data analysis needs of the global mitochondrial disease clinical and research community. A central Web portal (https://mseqdr.org) facilitates the coherent compilation, organization, annotation, and analysis of sequence data from both nuclear and mitochondrial genomes of individuals and families with suspected mitochondrial disease. This Web portal provides users with a flexible and expandable suite of resources to enable variant-, gene-, and exome-level sequence analysis in a secure, Web-based, and user-friendly fashion. Users can also elect to share data with other MSeqDR Consortium members, or even the general public, either by custom annotation tracks or through use of a convenient distributed annotation system (DAS) mechanism. A range of data visualization and analysis tools are provided to facilitate user interrogation and understanding of genomic, and ultimately phenotypic, data of relevance to mitochondrial biology and disease. Currently available tools for nuclear and mitochondrial gene analyses include an MSeqDR GBrowse instance that hosts optimized mitochondrial disease and mitochondrial DNA (mtDNA) specific annotation tracks, as well as an MSeqDR locus-specific database (LSDB) that curates variant data on more than 1,300 genes that have been implicated in mitochondrial disease and/or encode mitochondria-localized proteins. MSeqDR is integrated with a diverse array of mtDNA data analysis tools that are both freestanding and incorporated into an online exome-level dataset curation and analysis resource (GEM.app) that is being optimized to support needs of the MSeqDR community. In addition, MSeqDR supports mitochondrial disease phenotyping and ontology tools, and provides variant pathogenicity assessment features that enable community review, feedback, and integration with the public ClinVar variant annotation resource. A centralized Web-based informed consent process is being developed, with implementation of a Global Unique Identifier (GUID) system to integrate data deposited on a given individual from different sources. Community-based data deposition into MSeqDR has already begun. Future efforts will enhance capabilities to incorporate phenotypic data that enhance genomic data analyses. MSeqDR will fill the existing void in bioinformatics tools and centralized knowledge that are necessary to enable efficient nuclear and mtDNA genomic data interpretation by a range of shareholders across bo...

BackgroundMitochondrial disorders are difficult to diagnose due to extreme genetic and phenotypic heterogeneities.MethodsWe explored the utility of targeted next-generation sequencing for the diagnosis of mitochondrial disorders in 148 patients submitted for clinical testing. A panel of 447 nuclear genes encoding mitochondrial respiratory chain complexes, and other genes inducing secondary mitochondrial dysfunction or that cause diseases which mimic mitochondrial disorders were tested.ResultsWe identified variants considered to be possibly disease-causing based on family segregation data and/or variants already known to cause disease in twelve genes in thirteen patients. Rare or novel variants of unknown significance were identified in 45 additional genes for various metabolic, genetic or neurogenetic disorders.ConclusionsPrimary mitochondrial defects were confirmed only in four patients indicating that majority of patients with suspected mitochondrial disorders are presumably not the result of direct impairment of energy production. Our results support that clinical and routine laboratory ascertainment for mitochondrial disorders are challenging due to significant overlapping non-specific clinical symptoms and lack of specific biomarkers. While next-generation sequencing shows promise for diagnosing suspected mitochondrial disorders, the challenges remain as the underlying genetic heterogeneity may be greater than suspected and it is further confounded by the similarity of symptoms with other conditions as we report here.

Incomplete malaria control efforts have resulted in a worldwide increase in resistance to drugs used to treat the disease. A complex array of mutations underlying antimalarial drug resistance complicates efficient monitoring of parasite populations and limits the success of malaria control efforts in regions of endemicity. To improve the surveillance of Plasmodium falciparum drug resistance, we developed a multiplex ligase detection reaction-fluorescent-microsphere-based assay (LDR-FMA) that identifies single nucleotide polymorphisms (SNPs) in the P. falciparum dhfr (9 alleles), dhps (10 alleles), and pfcrt (3 alleles) genes associated with resistance to Fansidar and chloroquine. We evaluated 1,121 blood samples from study participants in the Wosera region of Papua New Guinea, where malaria is endemic. Results showed that 468 samples were P. falciparum negative and 453 samples were P. falciparum positive by a Plasmodium species assay and all three gene assays (concordance, 82.2%). Plasmodium falciparum strains exhibit resistance to many antimalarial drugs in most regions of the world where malaria is endemic. In some regions, individual strains are resistant to more than one drug. With a very limited arsenal of safe and effective antimalarial drugs, complex genetic factors contributing to drug resistance pose a constant challenge to efforts to control this important human parasite. Moreover, it has been observed that as drug-resistant P. falciparum evolves and spreads within regions of endemicity and resistant strains become predominant, both transmission and the morbidity and mortality attributable to malaria increase (29). With increasing travel around the world, drug-resistant malaria parasites present further challenges in prescribing effective prophylactic treatment for tourists, military personnel, and humanitarian aid workers. Additionally, infected travelers are likely to increase the exchange of parasite strains between regions where different patterns of drug resistance are observed.Molecular genetic studies of P. falciparum have enabled identification of a number of specific mutations in genes linked to resistance to specific antimalarial drugs (34, 35). These include genes encoding the P. falciparum chloroquine resistance transporter (pfcrt) (10), dihydrofolate reductase (dhfr) (7, 24), and dihydropterate synthetase (dhps) (30, 31), which confer resistance to chloroquine, pyrimethamine, and sulfadoxine, respectively. Mutations in the latter two genes confer resistance to the drug combinations Fansidar (pyrimethaminesulfadoxine) and LAPDAP (chlorproguanil-dapsone) (22, 36). Our interest in monitoring these genes for single nucleotide polymorphisms (SNPs) associated with drug resistance was based on a number of technical and field surveillance objectives. Whereas numerous PCR-based approaches have been used to analyze polymorphisms in the P. falciparum dhps, dhfr, and pfcrt genes, most strategies involve post-PCR restriction fragment length polymorphism or DNA probe hybridization methods that are cumbe...