Steve Lefever

Molecular diagnostics for congenital hearing loss including 15 deafness genes using a next generation sequencing platform.

BackgroundHereditary hearing loss (HL) can originate from mutations in one of many genes involved in the complex process of hearing. Identification of the genetic defects in patients is currently labor intensive and expensive. While screening with Sanger sequencing for GJB2 mutations is common, this is not the case for the other known deafness genes (> 60). Next generation sequencing technology (NGS) has the potential to be much more cost efficient. Published methods mainly use hybridization based target enrichment procedures that are time saving and efficient, but lead to loss in sensitivity. In this study we used a semi-automated PCR amplification and NGS in order to combine high sensitivity, speed and cost efficiency.ResultsIn this proof of concept study, we screened 15 autosomal recessive deafness genes in 5 patients with congenital genetic deafness. 646 specific primer pairs for all exons and most of the UTR of the 15 selected genes were designed using primerXL. Using patient specific identifiers, all amplicons were pooled and analyzed using the Roche 454 NGS technology. Three of these patients are members of families in which a region of interest has previously been characterized by linkage studies. In these, we were able to identify two new mutations in CDH23 and OTOF. For another patient, the etiology of deafness was unclear, and no causal mutation was found. In a fifth patient, included as a positive control, we could confirm a known mutation in TMC1.ConclusionsWe have developed an assay that holds great promise as a tool for screening patients with familial autosomal recessive nonsyndromal hearing loss (ARNSHL). For the first time, an efficient, reliable and cost effective genetic test, based on PCR enrichment, for newborns with undiagnosed deafness is available.

Practical Tools to Implement Massive Parallel Pyrosequencing of PCR Products in Next Generation Molecular Diagnostics

Despite improvements in terms of sequence quality and price per basepair, Sanger sequencing remains restricted to screening of individual disease genes. The development of massively parallel sequencing (MPS) technologies heralded an era in which molecular diagnostics for multigenic disorders becomes reality. Here, we outline different PCR amplification based strategies for the screening of a multitude of genes in a patient cohort. We performed a thorough evaluation in terms of set-up, coverage and sequencing variants on the data of 10 GS-FLX experiments (over 200 patients). Crucially, we determined the actual coverage that is required for reliable diagnostic results using MPS, and provide a tool to calculate the number of patients that can be screened in a single run. Finally, we provide an overview of factors contributing to false negative or false positive mutation calls and suggest ways to maximize sensitivity and specificity, both important in a routine setting. By describing practical strategies for screening of multigenic disorders in a multitude of samples and providing answers to questions about minimum required coverage, the number of patients that can be screened in a single run and the factors that may affect sensitivity and specificity we hope to facilitate the implementation of MPS technology in molecular diagnostics.

Massively parallel sequencing for early molecular diagnosis in Leber congenital amaurosis

Purpose:Leber congenital amaurosis (LCA) is a rare congenital retinal dystrophy associated with 16 genes. Recent breakthroughs in LCA gene therapy offer the first prospect of treating inherited blindness, which requires an unequivocal and early molecular diagnosis. While present genetic tests do not address this due to a tremendous genetic heterogeneity, massively parallel sequencing (MPS) strategies might bring a solution. Here, we developed a comprehensive molecular test for LCA based on targeted MPS of all exons of 16 known LCA genes.Methods:We designed a unique and flexible workflow for targeted resequencing of all 236 exons from 16 LCA genes based on quantitative PCR (qPCR) amplicon ligation, shearing, and parallel sequencing of multiple patients on a single lane of a short-read sequencer. Twenty-two prescreened LCA patients were included, five of whom had a known molecular cause.Results:Validation of 107 variations was performed as proof of concept. In addition, the causal genetic defect and a single heterozygous mutation were identified in 3 and 5, respectively, of 17 patients without previously identified mutations.Conclusion:We propose a novel targeted MPS-based approach that is suitable for accurate, fast, and cost-effective early molecular testing in LCA, and easily applicable in other genetic disorders.Genet Med 2012:14(6):576–585

Identity-by-descent–guided mutation analysis and exome sequencing in consanguineous families reveals unusual clinical and molecular findings in retinal dystrophy

Purpose:Autosomal recessive retinal dystrophies are clinically and genetically heterogeneous, which hampers molecular diagnosis. We evaluated identity-by-descent–guided Sanger sequencing or whole-exome sequencing in 26 families with nonsyndromic (19) or syndromic (7) autosomal recessive retinal dystrophies to identify disease-causing mutations.Methods:Patients underwent genome-wide identity-by-descent mapping followed by Sanger sequencing (16) or whole-exome sequencing (10). Whole-exome sequencing data were filtered against identity-by-descent regions and known retinal dystrophy genes. The medical history was reviewed in mutation-positive families.Results:We identified mutations in 14 known retinal dystrophy genes in 20/26 (77%) families: ABCA4, CERKL, CLN3, CNNM4, C2orf71, IQCB1, LRAT, MERTK, NMNAT1, PCDH15, PDE6B, RDH12, RPGRIP1, and USH2A. Whole-exome sequencing in single individuals revealed mutations in either the largest or smaller identity-by-descent regions, and a compound heterozygous genotype in NMNAT1. Moreover, a novel deletion was found in PCDH15. In addition, we identified mutations in CLN3, CNNM4, and IQCB1 in patients initially diagnosed with nonsyndromic retinal dystrophies.Conclusion:Our study emphasized that identity-by-descent–guided mutation analysis and/or whole-exome sequencing are powerful tools for the molecular diagnosis of retinal dystrophy. Our approach uncovered unusual molecular findings and unmasked syndromic retinal dystrophies, guiding future medical management. Finally, elucidating ABCA4, LRAT, and MERTK mutations offers potential gene-specific therapeutic perspectives.Genet Med 16 9, 671–680.

Regulatory microRNA Network Identification in Bovine Blastocyst Development

Mammalian blastocyst formation is characterized by two lineage segregations resulting in the formation of the trophectoderm, the hypoblast, and the epiblast cell lineages. Cell fate determination during these early lineage segregations is associated with changes in the expression of specific transcription factors. In addition to the transcription factor-based control, it has become clear that also microRNAs (miRNAs) play an important role in the post-transcriptional regulation of pluripotency and differentiation. To elucidate the role of miRNAs in early lineage segregation, we compared the miRNA expression in early bovine blastocysts with the more advanced stage of hatched blastocysts. Reverse transcription-quantitative PCR-based miRNA expression profiling revealed eight upregulated miRNAs (miR-127, miR-130a, miR-155, miR-196a, miR-203, miR-28, miR-29c, and miR-376a) and four downregulated miRNAs (miR-135a, miR-218, miR-335, and miR-449b) in hatched blastocysts. Through an integrative analysis of matching miRNA and mRNA expression data, candidate miRNA-mRNA interaction pairs were prioritized for validation. Using an in vitro luciferase reporter assay, we confirmed a direct interaction between miR-218 and CDH2, miR-218 and NANOG, and miR-449b and NOTCH1. By interfering with the FGF signaling pathway, we found functional evidence that miR-218, mainly expressed in the inner cell mass, regulates the NANOG expression in the bovine blastocyst in response to FGF signaling. The results of this study expand our knowledge about the miRNA signature of the bovine blastocyst and of the interactions between miRNAs and cell fate regulating transcription factors.

Flexible, Scalable, and Efficient Targeted Resequencing on a Benchtop Sequencer for Variant Detection in Clinical Practice

The release of benchtop next‐generation sequencing (NGS) instruments has paved the way to implement the technology in clinical setting. The need for flexible, qualitative, and cost‐efficient workflows is high. We used singleplex‐PCR for highly efficient target enrichment, allowing us to reach the quality standards set in Sanger sequencing‐based diagnostics. For the library preparation, a modified NexteraXT protocol was used, followed by sequencing on a MiSeq instrument. With an innovative pooling strategy, high flexibility, scalability, and cost‐efficiency were obtained, independent of the availability of commercial kits. The approach was validated for ∼250 genes associated with monogenic disorders. An overall sensitivity (>99%) similar to Sanger sequencing was observed in combination with a positive predictive value of >98%. The distribution of coverage was highly uniform, guaranteeing a minimal number of gaps to be filled with alternative methods. ISO15189‐accreditation was obtained for the workflow. A major asset of the singleplex PCR‐based enrichment is that new targets can be easily implemented. Diagnostic laboratories have validated assays available ensuring that the proposed workflow can easily be adopted. Although our platform was optimized for constitutional variant detection of monogenic disease genes, it is now also used as a model for somatic mutation detection in acquired diseases.

Expressed Repeat Elements Improve RT-qPCR Normalization across a Wide Range of Zebrafish Gene Expression Studies

The selection and validation of stably expressed reference genes is a critical issue for proper RT-qPCR data normalization. In zebrafish expression studies, many commonly used reference genes are not generally applicable given their variability in expression levels under a variety of experimental conditions. Inappropriate use of these reference genes may lead to false interpretation of expression data and unreliable conclusions. In this study, we evaluated a novel normalization method in zebrafish using expressed repetitive elements (ERE) as reference targets, instead of specific protein coding mRNA targets. We assessed and compared the expression stability of a number of EREs to that of commonly used zebrafish reference genes in a diverse set of experimental conditions including a developmental time series, a set of different organs from adult fish and different treatments of zebrafish embryos including morpholino injections and administration of chemicals. Using geNorm and rank aggregation analysis we demonstrated that EREs have a higher overall expression stability compared to the commonly used reference genes. Moreover, we propose a limited set of ERE reference targets (hatn10, dna15ta1 and loopern4), that show stable expression throughout the wide range of experiments in this study, as strong candidates for inclusion as reference targets for qPCR normalization in future zebrafish expression studies. Our applied strategy to find and evaluate candidate expressed repeat elements for RT-qPCR data normalization has high potential to be used also for other species.

Differential expression of lncRNAs during the HIV replication cycle: an underestimated layer in the HIV-host interplay

Studying the effects of HIV infection on the host transcriptome has typically focused on protein-coding genes. However, recent advances in the field of RNA sequencing revealed that long non-coding RNAs (lncRNAs) add an extensive additional layer to the cell’s molecular network. Here, we performed transcriptome profiling throughout a primary HIV infection in vitro to investigate lncRNA expression at the different HIV replication cycle processes (reverse transcription, integration and particle production). Subsequently, guilt-by-association, transcription factor and co-expression analysis were performed to infer biological roles for the lncRNAs identified in the HIV-host interplay. Many lncRNAs were suggested to play a role in mechanisms relying on proteasomal and ubiquitination pathways, apoptosis, DNA damage responses and cell cycle regulation. Through transcription factor binding analysis, we found that lncRNAs display a distinct transcriptional regulation profile as compared to protein coding mRNAs, suggesting that mRNAs and lncRNAs are independently modulated. In addition, we identified five differentially expressed lncRNA-mRNA pairs with mRNA involvement in HIV pathogenesis with possible cis regulatory lncRNAs that control nearby mRNA expression and function. Altogether, the present study demonstrates that lncRNAs add a new dimension to the HIV-host interplay and should be further investigated as they may represent targets for controlling HIV replication.

RDML-Ninja and RDMLdb for standardized exchange of qPCR data

BackgroundThe universal qPCR data exchange file format RDML is today well accepted by the scientific community, part of the MIQE guidelines and implemented in many qPCR instruments. With the increased use of RDML new challenges emerge. The flexibility of the RDML format resulted in some implementations that did not meet the expectations of the consortium in the level of support or the use of elements.ResultsIn the current RDML version 1.2 the description of the elements was sharpened. The open source editor RDML-Ninja was released (http://sourceforge.net/projects/qpcr-ninja/). RDML-Ninja allows to visualize, edit and validate RDML files and thus clarifies the use of RDML elements. Furthermore RDML-Ninja serves as reference implementation for RDML and enables migration between RDML versions independent of the instrument software. The database RDMLdb will serve as an online repository for RDML files and facilitate the exchange of RDML data (http://www.rdmldb.org). Authors can upload their RDML files and reference them in publications by the unique identifier provided by RDMLdb. The MIQE guidelines propose a rich set of information required to document each qPCR run. RDML provides the vehicle to store and maintain this information and current development aims at further integration of MIQE requirements into the RDML format.ConclusionsThe editor RDML-Ninja and the database RDMLdb enable scientists to evaluate and exchange qPCR data in the instrument-independent RDML format. We are confident that this infrastructure will build the foundation for standardized qPCR data exchange among scientists, research groups, and during publication.

How long noncoding RNAs enforce their will on mitochondrial activity : regulation of mitochondrial respiration, reactive oxygen species production, apoptosis, and metabolic reprogramming in cancer

The cellular transcriptome contains a wide diversity of untranslated RNAs, of which the class of regulatory long noncoding RNAs (lncRNAs) has only recently been recognized. Evidence swiftly accumulates of lncRNAs influencing mitochondrial activities of eukaryotic cells, and perturbed expression is conspicuously associated with human diseases. In this review, we describe the multifaceted effects of lncRNAs on mitochondrial function, more particularly on the balance between oxidative phosphorylation and glycolysis, on the production of reactive oxygen species, and on apoptosis in human cells. Emphasis is placed on the involvement of lncRNAs in cancer metabolism, as tumor cells rely heavily on modifications of mitochondrial functioning as an essential component for sustained tumorigenesis and cancer progression. From the nonexhaustive list of lncRNAS described in this review, ANRIL, AScmtRNA, H19, HOTAIR, LincRNA-p21, MALAT1, RMRP, SAMMSON, and VL30 have emerged as potent regulators of mitochondrial metabolism. Due to their key role in cancer progression, they represent potential targets of innovative lncRNA-based treatment strategies.