Participant-driven matchmaking in the genomic era.

Whole-genome and whole-exome sequencing are increasingly useful diagnostic tools for novel monogenic conditions. In order to confirm diagnoses made using these technologies, genomic matchmaking-the matching of cases with similar phenotypic and/or genotypic profiles, to narrow the number of candidate genes or ascertain a condition's etiology with greater certainty-is essential. Yet, due to current limitations on the size of matchmaking networks and data sets available to support them, identifying a match can be difficult. We argue that matchmaking efforts led by affected individuals and their families-participant-led efforts-offer a twofold solution to this need, in that participants both have the capacity to access larger networks and to provide more detailed sets of phenotypic and genotypic data. These features of participant-led efforts have the potential to increase the value of matchmaking networks, both in terms of number of matches and in terms of the overall energy of the network. We provide two examples of participant-led matchmaking, and propose a model for scaling these efforts.

Mitochondrial Disease Sequence Data Resource (MSeqDR): a global grass-roots consortium to facilitate deposition, curation, annotation, and integrated analysis of genomic data for the mitochondrial disease clinical and research communities.

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 the 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 1300 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 both clinical diagnostic and research settings. Ultimately, MSeqDR is focused on empowering the global mitochondrial disease community to better define and explore mitochondrial diseases.

Broadband optical absorbance spectroscopy using a whispering gallery mode microsphere resonator.

We demonstrate the ability to excite and monitor many whispering gallery modes (WGMs) of a microsphere resonator simultaneously in order to make broadband optical absorbance measurements. The 340 microm diameter microsphere is placed in a microfluidic channel. A hemispherical prism is used for coupling the WGMs into and out of the microsphere. The flat surface of the prism seals the microfluidic channel. The slight nonsphericity in the microsphere results in coupling to precessed modes whose emission is spatially separated from the reflected excitation light. The evanescent fields of the light trapped in WGMs interact with the surrounding environment. The change in transmission observed in the precessed modes is used to determine the absorbance of the surrounding environment. In contrast to our broadband optical absorbance measurements, previous WGM sensors have used only a single narrow mode to measure properties such as refractive index. With the microfluidic cell, we have measured the absorbance of solutions of dyes (lissamine green B, sunset yellow, orange G, and methylene blue), aromatic molecules (benzylamine and benzoic acid), and biological molecules (tryptophan, phenylalanine, tyrosine, and o-phospho-L-tyrosine) at visible and ultraviolet wavelengths. The microsphere surface was reacted with organosilane molecules to attach octadecyl groups, amino groups, and fluorogroups to the surface. Both electrostatic and hydrophobic interactions were observed between the analytes and the microsphere surface, as indicated by changes in the measured effective pathlength with different organosilanes. For a given analyte and coated microsphere, the pathlength measurement was repeatable within a few percent. Methylene blue dye had a very strong interaction with the surface and pathlengths of several centimeters were measured. Choosing an appropriate surface coating to interact with a specific analyte should result in the highest sensitivity detection.

Creating a data resource: what will it take to build a medical information commons?

National and international public-private partnerships, consortia, and government initiatives are underway to collect and share genomic, personal, and healthcare data on a massive scale. Ideally, these efforts will contribute to the creation of a medical information commons (MIC), a comprehensive data resource that is widely available for both research and clinical uses. Stakeholder participation is essential in clarifying goals, deepening understanding of areas of complexity, and addressing long-standing policy concerns such as privacy and security and data ownership. This article describes eight core principles proposed by a diverse group of expert stakeholders to guide the formation of a successful, sustainable MIC. These principles promote formation of an ethically sound, inclusive, participant-centric MIC and provide a framework for advancing the policy response to data-sharing opportunities and challenges.

Separating human DNA mixtures using denaturing high-performance liquid chromatography.

DNA mixtures represent challenging samples that are rarely amenable to direct DNA sequence analysis and many of the strategies available to separate mixtures are both labor and time intensive. Denaturing high-performance liquid chromatography is an accurate and rapid approach for the detection and scoring of mutations. It can also be used to separate DNA mixtures. The technique relies on the chromatographic separation of crosshybridization products to isolate the individual components of a mixture. By eliminating secondary amplification and excessive manipulation prior to sequencing, denaturing high-performance liquid chromatography can streamline the analysis of conditions ranging from somatic mosaicism, microchimerism and mitochondrial heteroplasmy to evidentiary material containing mixtures of DNA encountered in forensic investigations.

Testing an online, dynamic consent portal for large population biobank research.

BACKGROUND: Michigan's BioTrust for Health, a public health research biobank comprised of residual dried bloodspot (DBS) cards from newborn screening contains over 4 million samples collected without written consent. Participant-centric initiatives are IT tools that hold great promise to address the consent challenges in biobank research. METHODS: Working with Private Access Inc., a pioneer in patient-centric web solutions, we created and pilot tested a dynamic informed consent simulation, paired with an educational website, focusing on consent for research utilizing DBSs in Michigan's BioTrust for Health. RESULTS: Out of 187 pilot testers recruited in 2 groups, 137 completed the consent simulation and exit survey. Over 50% indicated their willingness to set up an account if the simulation went live and to recommend it to others. Participants raised concerns about the process of identity verification and appeared to have little experience with sharing health information online. CONCLUSIONS: Applying online, dynamic approaches to address the consent challenges raised by biobanks with legacy sample collections should be explored, given the positive reaction to our pilot test and the strong preference for active consent. Balancing security and privacy with accessibility and ease of use will continue to be a challenge.

Human Immunodeficiency Virus nef signature sequences are associated with pulmonary hypertension.

Severe pulmonary hypertension (PH) associated with vascular remodeling is a long-term complication of HIV infection (HIV-PH) affecting 1/200 infected individuals vs. 1/200,000 frequency in the uninfected population. Factors accounting for increased PH susceptibility in HIV-infected individuals are unknown. Rhesus macaques infected with chimeric SHIVnef virions but not with SIV display PH-like pulmonary vascular remodeling suggesting that HIV-Nef is associated with PH; these monkeys showed changes in nef sequences that correlated with pathogenesis after passage in vivo. We further examined whether HIV-nef alleles in HIV-PH subjects have signature sequences associated with the disease phenotype. We evaluated specimens from participants with and without HIV-PH from European Registries and validated results with samples collected as part of the Lung-HIV Studies in San Francisco. We found that 10 polymorphisms in nef were overrepresented in blood cells or lung tissue specimens from European HIV-PH individuals but significantly less frequent in HIV-infected individuals without PH. These polymorphisms mapped to known functional domains in Nef. In the validation cohort, 7/10 polymorphisms in the HIV-nef gene were confirmed; these polymorphisms arose independently from viral load, CD4(+) T cell counts, length of infection, and antiretroviral therapy status. Two out of 10 polymorphisms were previously reported in macaques with PH-like pulmonary vascular remodeling. Cloned recombinant Nef proteins from clinical samples down-regulated CD4, suggesting that these primary isolates are functional. This study offers new insights into the association between Nef polymorphisms in functional domains and the HIV-PH phenotype. The utility of these polymorphisms as predictors of PH should be examined in a larger population.

Electronic consent channels: preserving patient privacy without handcuffing researchers.

Advances in health information technology and electronic medical records have the tremendous potential to accelerate translational and clinical research. However, privacy concerns threaten to be a rate-limiting factor. By recognizing and responding to patient privacy concerns, policy-makers, researchers, and information technology leaders have the opportunity to transform trial recruitment and make it safer to electronically locate and convey sensitive health information.

Forensic utility of mitochondrial DNA analysis based on denaturing high-performance liquid chromatography.

AIM: To determine the forensic utility for pairwise DNA comparisons and DNA mixture resolution with denaturing high-performance liquid chromatography (DHPLC) of human mitochondrial DNA (mtDNA). METHODS: MtDNA hypervariable regions (HV) 1 and 2 from the mtDNA D-loop were amplified by the polymerase chain reaction and mixed between known and unknown sample sources. The DNA mixtures were denatured and reannealed, and the resultant homo- and heteroduplices were evaluated by temperature-modulated heteroduplex analysis by the DHPLC method. RESULTS: All 144 pairwise comparisons of HV1 and HV2 mtDNA fragments were successfully resolved by the DHPLC method. Forensic proficiency test standards were successfully resolved and DHPLC match/non-match results agreed with sequencing results provided by the test providers. The DHPLC method successfully identified one questioned sample that was prepared by the test provider as a body fluid mixture. MtDNA amplicon mixtures could be separated into their constitutive components by DHPLC and fraction collection approaches. CONCLUSIONS: DHPLC methods provide the forensic scientist with a powerful tool to rapidly screen mtDNA and may result in standardized methods to resolve mtDNA mixtures. These advances will allow mtDNA analysis in cases not previously examined by current sequencing-based approaches and could allow more forensic case samples to be entered into the proposed mtDNA Combined DNA Index System (CODIS trade mark ) databank as a result of mtDNA mixture resolution.

Clinical applications of denaturing high-performance liquid chromatography-based genotyping.

AIM: To develop and evaluate heteroduplex forming templates (HFTs) as a common set of molecular standards for genotyping by denaturing high-performance liquid chromatography (DHPLC) using hypervariable regions of human mitochondrial DNA (mtDNA) as a model system. METHODS: Hypervariable regions 1 and 2 from the mtDNA D-loop of 22 maternally related and unrelated human volunteers were amplified by polymerase chain reaction (PCR) and individually mixed with each of three HFTs. Following denaturation and reannealing of the mixture, the resulting hetero- and homoduplicies were separated by DHPLC using temperature-modulated heteroduplex analysis. RESULTS: Each of three HFTs, when cross-hybridized with a target mtDNA amplicon, induced the formation of an assemblage hetero- and homoduplex peaks, which were uniquely characteristic of a given mtDNA sequence variant. The mtDNA DHPLC profiles obtained in the current study were identical between maternal relatives and different between unrelated individuals--consistent with uniparental maternal inheritance of mtDNA in humans. CONCLUSION: DHPLC in combination with a common set of HFTs targeted to a locus of interest can be used as a reliable means of genotyping. DHPLC profiles can be readily stored as a bit-coded string of hetero- and homoduplex peak retention times to form a searchable database. This approach to DHPLC genotyping will have immediate utility in extended pedigree analyses, where it will allow rapid sorting and/or confirmation of maternal lineages. Additional applications of DHPLC profiling include the discovery and scoring of clinically relevant nuclear and mitochondrial loci.

Subfemtosecond timing jitter between two independent, actively synchronized, mode-locked lasers.

With the implementation of a fast-bandwidth servo, along with improved laser construction and associated better passive stability, we have achieved subfemtosecond relative timing jitter between two independent, actively synchronized, mode-locked Ti:sapphire lasers. Timing jitter of 0.58 fs is obtained with a 160-Hz observation bandwidth over several seconds. Within a 2-MHz observation bandwidth, the timing jitter is 1.75 fs. Excellent repeatability and rapid speed in setting an arbitrary time delay between two pulses are also demonstrated.