Emily M. Coonrod

The role of Fis1p–Mdv1p interactions in mitochondrial fission complex assembly

Mitochondrial division requires coordinated interactions among Fis1p, Mdv1p, and the Dnm1p GTPase, which assemble into fission complexes on the outer mitochondrial membrane. The integral outer membrane protein Fis1p contains a cytoplasmic domain consisting of a tetratricopeptide repeat (TPR)–like fold and a short NH2-terminal helix. Although it is known that the cytoplasmic domain is necessary for assembly of Mdv1p and Dnm1p into fission complexes, the molecular details of this assembly are not clear. In this study, we provide new evidence that the Fis1p–Mdv1p interaction is direct. Furthermore, we show that conditional mutations in the Fis1p TPR-like domain cause fission complex assembly defects that are suppressed by mutations in the Mdv1p-predicted coiled coil. We also define separable functions for the Fis1p NH2-terminal arm and TPR-like fold. These studies suggest that the concave binding surface of the Fis1p TPR-like fold interacts with Mdv1p during mitochondrial fission and that Mdv1p facilitates Dnm1p recruitment into functional fission complexes.

Ugo1p Is a Multipass Transmembrane Protein with a Single Carrier Domain Required for Mitochondrial Fusion

The outer mitochondrial membrane protein Ugo1 forms a complex with the Fzo1p and Mgm1p GTPases that regulates mitochondrial fusion in yeast. Ugo1p contains two putative carrier domains (PCDs) found in mitochondrial carrier proteins (MCPs). Mitochondrial carrier proteins are multipass transmembrane proteins that actively transport molecules across the inner mitochondrial membrane. Mitochondrial carrier protein transport requires functional carrier domains with the consensus sequence PX(D/E)XX(K/R). Mutation of charged residues in this consensus sequence disrupts transport function. In this study, we used targeted mutagenesis to show that charge reversal mutations in Ugo1p PCD2, but not PCD1, disrupt mitochondrial fusion. Ugo1p is reported to be a single‐pass transmembrane protein despite the fact that it contains several additional predicted transmembrane segments. Using a combination of protein targeting and membrane extraction experiments, we provide evidence that Ugo1p contains additional transmembrane domains and is likely a multipass transmembrane protein. These studies identify PCD2 as a functional domain of Ugo1p and provide the first experimental evidence for a multipass topology of this essential fusion component.

Clinical analysis of genome next-generation sequencing data using the Omicia platform

Aims: Next-generation sequencing is being implemented in the clinical laboratory environment for the purposes of candidate causal variant discovery in patients affected with a variety of genetic disorders. The successful implementation of this technology for diagnosing genetic disorders requires a rapid, user-friendly method to annotate variants and generate short lists of clinically relevant variants of interest. This report describes Omicia’s Opal platform, a new software tool designed for variant discovery and interpretation in a clinical laboratory environment. The software allows clinical scientists to process, analyze, interpret and report on personal genome files. Materials & Methods: To demonstrate the software, the authors describe the interactive use of the system for the rapid discovery of disease-causing variants using three cases. Results & Conclusion: Here, the authors show the features of the Opal system and their use in uncovering variants of clinical significance.

Next-generation sequencing of custom amplicons to improve coverage of HaloPlex multigene panels

Next-generation sequencing (NGS) of multigene panels performed for genetic clinical diagnostics requires 100% coverage of all targeted genes. In the genetic diagnostics laboratory, coverage gaps are typically filled with Sanger sequencing after NGS data are collected and analyzed. Libraries prepared using the hybridization-based custom capture HaloPlex method are covered at ~98% and include gaps in coverage because of the location of the restriction enzyme sites used for fragmentation and differences in the designed and actual library insert size. We describe a method for improving the coverage of HaloPlex libraries by generating a set of amplicons spanning known low-coverage regions that are pooled, indexed by sample, and sequenced together with the HaloPlex libraries. This approach reduces the number of post-NGS Sanger sequencing reactions required and complements any NGS library preparation method when complete gene coverage is necessary.

The Yeast vps Class E Mutants: The Beginning of the Molecular Genetic Analysis of Multivesicular Body Biogenesis

In 1992, Raymond et al. published a compilation of the 41 yeast vacuolar protein sorting (vps) mutant groups and described a large class of mutants (class E vps mutants) that accumulated an exaggerated prevacuolar endosome-like compartment. Further analysis revealed that this “class E compartment” contained soluble vacuolar hydrolases, vacuolar membrane proteins, and Golgi membrane proteins unable to recycle back to the Golgi complex, yet these class E vps mutants had what seemed to be normal vacuoles. The 13 class E VPS genes were later shown to encode the proteins that make up the complexes required for formation of intralumenal vesicles in late endosomal compartments called multivesicular bodies, and for the sorting of ubiquitinated cargo proteins into these internal vesicles for eventual delivery to the vacuole or lysosome.

Arabidopsis has two functional orthologs of the yeast V-ATPase assembly factor Vma21p.

How individual protein subunits assemble into the higher order structure of a protein complex is not well understood. Four proteins dedicated to the assembly of the V0 subcomplex of the V‐adenosine triphosphatase (V‐ATPase) in the endoplasmic reticulum (ER) have been identified in yeast, but their precise mode of molecular action remains to be identified. In contrast to the highly conserved subunits of the V‐ATPase, orthologs of the yeast assembly factors are not easily identified based on sequence similarity. We show in this study that two ER‐localized Arabidopsis proteins that share only 25% sequence identity with Vma21p can functionally replace this yeast assembly factor. Loss of AtVMA21a function in RNA interference seedlings caused impaired cell expansion and changes in Golgi morphology characteristic for plants with reduced V‐ATPase activity, and we therefore conclude that AtVMA21a is the first V‐ATPase assembly factor identified in a multicellular eukaryote. Moreover, VMA21p acts as a dedicated ER escort chaperone, a class of substrate‐specific accessory proteins so far not identified in higher plants.

Translating exome sequencing from research to clinical diagnostics

Abstract In the relatively short time frame since the introduction of next generation sequencing, it has become a method of choice for complex genomic research studies. As a paradigm shifting technology, we are now witnessing its translation into clinical diagnostic laboratories for patient care. Multi-gene panels for a variety of disorders are now available in several clinical laboratories based on targeted gene enrichment followed by next generation sequencing. Genome wide interrogation of protein coding regions, or exome sequencing, has been successfully and increasingly applied in the research setting for the elucidation of candidate genes and causal variants in individuals and families with a diversity of rare and complex genetic disorders. Based on this progress, exome sequencing is also beginning a translational process into clinical practice. However, introducing exome sequencing as a diagnostic modality poses new technical and bioinformatics challenges for clinical laboratories. In this review, we present technical and bioinformatics aspects of exome sequencing, describe representative examples from the literature of how exome sequencing has been used for candidate gene discovery, and discuss considerations for its clinical translation.

Report on the effects of fragment size, indexing, and read length on HLA sequencing on the Illumina MiSeq

Single-molecule sequencing should allow for unambiguous, accurate, and high-throughput HLA typing. In this proof of principle study, we investigated the effects of fragment size for library preparation, indexing strategy, and read length on HLA typing. Whole gene amplicons of HLA-A, B, C, DRB1, and DQB1 were obtained by long-range PCR. For library preparation, two fragment sizes were evaluated: 100-300bp and 300-600bp. For sample multiplexing, two indexing strategies were compared: indexing-by-amplicon, where each individual amplicon is barcoded, and indexing-by-patient, where each patients five loci are equimolarly pooled after PCR and indexed with the same barcode. Sequencing was performed on an Illumina MiSeq instrument using paired-end 150bp and 250bp read lengths. Our results revealed that the 300-600bp fragments in the 2×250 MiSeq group gave the most accurate sequencing results. There was no difference in HLA typing results between the two indexing strategies, suggesting that indexing-by-patient, which is much simpler, is a viable option. In conclusion, enzymatic fragmentation of pooled whole gene amplicons is a suitable strategy for HLA typing by next-generation sequencing on the Illumina MiSeq.

Next-Generation Sequencing: Principles for Clinical Application

High-throughput, massively parallel DNA sequencing, more commonly termed “next-generation sequencing,” is an innovation in sequencing that emerged during the past decade. Next-generation sequencing (NGS) is not a single technology, but rather several different technologies that share a common feature of massively parallel sequencing of clonally amplified or single DNA molecules in a flow cell or chip. Inherent to NGS technologies are unique sequencing chemistries that differ from the Sanger dideoxynucleotide chain termination chemistry. NGS can generate, in a single instrument run, hundreds of millions to gigabases of nucleotide sequence data depending upon platform configuration, chemistry, and flow cell or chip capacity. This chapter describes principles of NGS and considerations for its application to clinical molecular tests. Although several NGS technologies have been commercialized, technologies finding greatest adoption into clinical laboratories are emphasized. Current clinical testing applications including multigene panels and exome and genome sequencing for candidate and causal gene identification are discussed. While the examples are primarily based on analyses for inherited disorders, the principles described are applicable to oncology and infectious diseases, with certain modifications mostly specific to the specimen characteristics and sensitivity requirements for these other applications.