How to identify pathogenic mutations among all those variations: Variant annotation and filtration in the genome sequencing era

High-throughput sequencing technologies have become fundamental for the identification of disease-causing mutations in human genetic diseases both in research and clinical testing contexts. The cumulative number of genes linked to rare diseases is now close to 3,500 with more than 1,000 genes identified between 2010 and 2014 because of the early adoption of Exome Sequencing technologies. However, despite these encouraging figures, the success rate of clinical exome diagnosis remains low due to several factors including wrong variant annotation and nonoptimal filtration practices, which may lead to misinterpretation of disease-causing mutations. In this review, we describe the critical steps of variant annotation and filtration processes to highlight a handful of potential disease-causing mutations for downstream analysis. We report the key annotation elements to gather at multiple levels for each mutation, and which systems are designed to help in collecting this mandatory information. We describe the filtration options, their efficiency, and limits and provide a generic filtration workflow and highlight potential pitfalls through a use case

Visual counts, bioacoustics and RADAR: three methods to study waterfowl prenuptial migration in Southern France

Conteos visuales, bioacústica y RADAR: tres métodos para estudiar la migración prenupcial de aves acuáticas en el sur de Francia Este estudio es el resultado de cuatro años de monitorización (2006–2009) en dos lugares durante la migración prenupcial. En cada uno, se llevó a cabo un seguimiento de 24 horas, durante periodos de 10 días, a lo largo de la segunda década de enero (J2), el mes de febrero (F1–F3), marzo (M1–M3) y la primera década de abril (A1). La migración se monitorizó mediante RADAR (FURUNO FAR2127), asociado con grabaciones bioacústicas nocturnas, y censos visuales en las mismas áreas. El esfuerzo de muestreo fue considerable: los conteos visuales totalizaron 282 conteos–sitios (n = 262.030 patos contados), mediante bioacústica se obtuvierono 9.573 vocalizaciones en 814 horas de grabación nocturna y mediante RADAR se registraron 67.368 ecos durante 2.128 horas de vigilancia. Los censos visuales muestran una disminución del número de aves a finales de enero/principios de febrero. Los registros nocturnos presentan un máximo o mínimo del índice bioacústico en F2 y F3 función del año. El RADAR, el mejor método para estudiar los movimientos de aves a nivel de población, identificó dos picos de abundancia diferentes, utilizando las variables “altura de vuelo > 400m” y “dirección de vuelo hacia noreste/sureste” consideradas como características de la migración prenupcial. El primer pico se detectó en F1 en el Sitio 1 sólo en el 2007 (un año de cada cuatro) y en F2 en el Sitio 2 sólo en el 2006 (un año de cada cuatro). Un segundo pico, de mayor intensidad, se detectó en M1 (Sitio 1) y en M2 (Sitio 2). Aunque todos los métodos considerados pueden tener sesgos, el uso de dos nuevas tecnologías en combinación con los conteos visuales, nos ha permitido obtener datos fiables y actuales sobre la migración de aves acuáticas en el área mediterránea.Conteos visuales, bioacústica y RADAR: tres métodos para estudiar la migración prenupcial de aves acuáticas en el sur de Francia Este estudio es el resultado de cuatro años de monitorización (2006–2009) en dos lugares durante la migración prenupcial. En cada uno, se llevó a cabo un seguimiento de 24 horas, durante periodos de 10 días, a lo largo de la segunda década de enero (J2), el mes de febrero (F1–F3), marzo (M1–M3) y la primera década de abril (A1). La migración se monitorizó mediante RADAR (FURUNO FAR2127), asociado con grabaciones bioacústicas nocturnas, y censos visuales en las mismas áreas. El esfuerzo de muestreo fue considerable: los conteos visuales totalizaron 282 conteos–sitios (n = 262.030 patos contados), mediante bioacústica se obtuvierono 9.573 vocalizaciones en 814 horas de grabación nocturna y mediante RADAR se registraron 67.368 ecos durante 2.128 horas de vigilancia. Los censos visuales muestran una disminución del número de aves a finales de enero/principios de febrero. Los registros nocturnos presentan un máximo o mínimo del índice bioacústico en F2 y F3 función del año. El RADAR, el mejor método para estudiar los movimientos de aves a nivel de población, identificó dos picos de abundancia diferentes, utilizando las variables “altura de vuelo > 400m” y “dirección de vuelo hacia noreste/sureste” consideradas como características de la migración prenupcial. El primer pico se detectó en F1 en el Sitio 1 sólo en el 2007 (un año de cada cuatro) y en F2 en el Sitio 2 sólo en el 2006 (un año de cada cuatro). Un segundo pico, de mayor intensidad, se detectó en M1 (Sitio 1) y en M2 (Sitio 2). Aunque todos los métodos considerados pueden tener sesgos, el uso de dos nuevas tecnologías en combinación con los conteos visuales, nos ha permitido obtener datos fiables y actuales sobre la migración de aves acuáticas en el área mediterránea.This study comes from four years (2006–2009) of monitoring on two sites during the prenuptial migration. On each site, a monitoring of 24 hours per each 10–day period from the second 10–day period of January (J2), though February (F1–F3) and March (M1–M3), up to the first 10–day period of April (A1). Monitoring was carried out by RADAR (FURUNO FAR2127), associated with nocturnal bioacoustics recordings, and visual censuses on the same areas. The monitoring effort was considerable: visual counts carried out represent 282 counts–sites (n = 262,030 ducks counted), bioacoustics detected 9,573 calls during 814 hours of nocturnal recording and RADAR recorded 67,368 echoes on a set of 2,128 hours of monitoring. Visual counts showed a decline in the number of birds from late January/early February. Two patterns were observed with the nocturnal recordings with a maximum or a minimum of the value of bioacoustics index on F2 and F3, depending on the years. RADAR, the most relevant method for tracking of bird movements at a population level, identified two different abundance peaks using variables ‘flight altitude > 400 m’ and ‘flight direction towards north–east/south–east’, considered as characteritics of the prenuptial migration. The first peak was detected during F1 on Site 1 only in 2007 (once every four years) and during F2 on Site 2 only in 2006 (once every four years). A second peak with a higher number of echoes was recorded on M1 (Site 1) and on M2 (Site 2). Although all methods may suffer from different biases, the combination of two new technologies complementary to visual counts provided reliable and updated data for waterfowl migration in the Mediterranean area

Landscape ecology and wild rabbit (Oryctolagus cuniculus) habitat modeling in the Mediterranean region

Ecología del paisaje y modelización del hábitat del conejo (Oryctolagus cuniculus) en la región mediterránea La modificación del paisaje es una de las razones de la disminución de las poblaciones de conejo. El objetivo de este estudio era la modelización del hábitat del conejo, usando la ecología del paisaje para crear un método de diagnóstico capaz de evaluar la calidad del hábitat a gran escala. Se observó la presencia/ausencia de conejos en 536 parcelas de 1 ha. Los recuentos nocturnos mediante transectos indicaron una abundancia relativa baja (IKA = 2,3 conejos/km). Por otra parte se realizó, mediante teledetección, una cartografía de uso del suelo con precisión métrica. Se identificaron el agua, el suelo desnudo, las plantas herbáceas, arbustos y los árboles. Se evaluaron la estructura del paisaje y su diversidad utilizando las variables disponibles en FRAGSTATS. Para evaluar la relación entre la presencia/ausencia del conejo y la estructura del paisaje se utilizó una regresión logística. Nuestros resultados indican que un hábitat adecuado tiene una gran diversidad, un número medio de parcelas y una pequeña proporción de arbustos. Estos resultados podrían ser utilizados para diagnosticar el paisaje antes de cualquier acción de gestión enfocada a aumentar las poblaciones de conejo, y a la inversa, ser un instrumento de control integrado en los casos de invasiones locales que causan daños a la agricultura.Ecología del paisaje y modelización del hábitat del conejo (Oryctolagus cuniculus) en la región mediterránea La modificación del paisaje es una de las razones de la disminución de las poblaciones de conejo. El objetivo de este estudio era la modelización del hábitat del conejo, usando la ecología del paisaje para crear un método de diagnóstico capaz de evaluar la calidad del hábitat a gran escala. Se observó la presencia/ausencia de conejos en 536 parcelas de 1 ha. Los recuentos nocturnos mediante transectos indicaron una abundancia relativa baja (IKA = 2,3 conejos/km). Por otra parte se realizó, mediante teledetección, una cartografía de uso del suelo con precisión métrica. Se identificaron el agua, el suelo desnudo, las plantas herbáceas, arbustos y los árboles. Se evaluaron la estructura del paisaje y su diversidad utilizando las variables disponibles en FRAGSTATS. Para evaluar la relación entre la presencia/ausencia del conejo y la estructura del paisaje se utilizó una regresión logística. Nuestros resultados indican que un hábitat adecuado tiene una gran diversidad, un número medio de parcelas y una pequeña proporción de arbustos. Estos resultados podrían ser utilizados para diagnosticar el paisaje antes de cualquier acción de gestión enfocada a aumentar las poblaciones de conejo, y a la inversa, ser un instrumento de control integrado en los casos de invasiones locales que causan daños a la agricultura.Landscape modification is one of the reasons for the decrease in rabbit populations. The objective of this study was to model wild rabbit habitat using landscape ecology to create a diagnosis method able to assess habitat quality at a large scale. Rabbit presence/absence was recorded on 536 plots of 1 ha. Spotlight transect counts indicated a low relative abundance (KIA = 2.3 rabbits/km). We produced a land use map with metric precision using remote sensing. Water, bare soil, herbaceous, shrubs and trees were identified. Landscape structure and diversity were evaluated using variables available in FRAGSTATS. A logistic regression was performed to assess the link between rabbit presence/absence and landscape structure. Our results indicate that a suitable habitat has a high diversity, a medium number of patches and a small proportion of shrubs. These results could be used to diagnose the landscape prior to any management action to enhance rabbit populations and conversely be helpful as a tool of integrated control in the cases of local outbreaks with agricultural damages

Large genomic rearrangements in the CFTR gene contribute to CBAVD

<p>Abstract</p> <p>Background</p> <p>By performing extensive scanning of whole coding and flanking sequences of the <it>CFTR (Cystic Fibrosis Transmembrane Conductance Regulator</it>) gene, we had previously identified point mutations in 167 out of 182 (91.7%) males with isolated congenital bilateral absence of the vas deferens (CBAVD). Conventional PCR-based methods of mutation analysis do not detect gross DNA lesions. In this study, we looked for large rearrangements within the whole <it>CFTR </it>locus in the 32 CBAVD patients with only one or no mutation.</p> <p>Methods</p> <p>We developed a semi-quantitative fluorescent PCR assay (SQF-PCR), which relies on the comparison of the fluorescent profiles of multiplex PCR fragments obtained from different DNA samples. We confirmed the gross alterations by junction fragment amplification and identified their breakpoints by direct sequencing.</p> <p>Results</p> <p>We detected two large genomic heterozygous deletions, one encompassing exon 2 (c.54-5811_c.164+2186del8108ins182) [or <it>CFTRdele2</it>], the other removing exons 22 to 24 (c.3964-3890_c.4443+3143del9454ins5) [or <it>CFTRdele 22_24</it>], in two males carrying a typical CBAVD mutation on the other parental <it>CFTR </it>allele. We present the first bioinformatic tool for exon phasing of the <it>CFTR </it>gene, which can help to rename the exons and the nomenclature of small mutations according to international recommendations and to predict the consequence of large rearrangements on the open reading frame.</p> <p>Conclusion</p> <p>Identification of large rearrangements further expands the <it>CFTR </it>mutational spectrum in CBAVD and should now be systematically investigated. We have designed a simple test to specifically detect the presence or absence of the two rearrangements identified in this study.</p

Knockout mice: Is it just genetics? Effect of enriched housing on fibulin-4+/- mice

Background. Fibulin-4 is an extracellular matrix protein expressed by vascular smooth muscle cells that is essential for maintaining arterial integrity. Fibulin-4-/- mice die just before birth due to arterial hemorrhage, but fibulin-4+/- mice appear to be outwardly normal. Experiments were therefore performed to determine whether fibulin-4+/- mice display arterial pathologies on a microscopic scale. After preliminary experiments were performed, a second purpose developed, which was to test the hypothesis that any observed pathologies would be ameliorated by housing the animals in enriched cages. Methodology. Fibulin-4+/- and wild-type mice were housed either four/cage in standard cages or two per cage in larger cages, each cage containing a tunnel and a wheel. After three weeks the mice were sacrificed, and the aortas perfusion-fixed and excised for light and electron microscopy. Principle Findings. When the mice were in standard cages, localized regions of disorganized extracellular matrix and collagen fibers consistently appeared between some of the medial smooth muscle cells in the fibulin-4+/- mice. In the wild-type mice, the smooth muscle cells were closely connected to each other and the media was more compact. The number of disorganized regions per square mm was significantly greater for fibulin-4+/- mice (172±43 (SEM)) than for wild-type mice (15±8) (p<0.01, n = 8). When the mice were in enriched cages, the fibulin-4+/- mice showed significantly fewer disorganized regions than those in standard cages (35±12) (p<0.05, n = 8). The wild type mice also showed fewer disorganized regions (3±2), but this difference was not significant. Conclusions. These results indicate that arterial pathologies manifested in fibulin-4+/- mice can be reduced by enriching the housing conditions, and imply that appropriate environments may counteract the effects of some genetic deficiencies

Broad phenotypic spectrum in familial adenomatous polyposis; from early onset and severe phenotypes to late onset of attenuated polyposis with the first manifestation at age 72

<p>Abstract</p> <p>Background</p> <p>Familial adenomatous polyposis (FAP) is typically characterized by multiple colonic polyps and frequent extracolonic features. Whereas the number of colonic polyps has been linked to the <it>APC </it>gene mutation, possible genotype-phenotype correlations largely remain to be defined for the extracolonic manifestations.</p> <p>Methods</p> <p>Full genomic sequencing combined with multiplex ligation-dependent probe amplification was used to identify <it>APC </it>gene mutations, which were correlated to the clinical presentations.</p> <p>Results</p> <p>10 novel <it>APC </it>gene mutations were identified in 11 families. A broad spectrum of extracolonic manifestations was identified in most of these individuals. Two sisters with an insertion in codon 528 (c.1582_1583insGC) both showed severe phenotypes with classical polyposis, upper gastrointestinal polyps and thyroid cancer. A woman with a 3'<it>APC </it>mutation (c.5030_5031insAA) developed colon cancer at age 72 as the first manifestation of attenuated FAP.</p> <p>Conclusion</p> <p>With an increasing number of FAP families diagnosed, a broad and variable tumor spectrum and a high frequency of extracolonic manifestations are gradually recognized. We report novel <it>APC </it>mutations and present two FAP cases that suggest familial aggregation of thyroid cancer and demonstrate the need to consider attenuated FAP also among elderly patients with colon cancer.</p

Antisense-induced exon skipping for duplications in Duchenne muscular dystrophy

<p>Abstract</p> <p>Background</p> <p>Antisense-mediated exon skipping is currently one of the most promising therapeutic approaches for Duchenne muscular dystrophy (DMD). Using antisense oligonucleotides (AONs) targeting specific exons the DMD reading frame is restored and partially functional dystrophins are produced. Following proof of concept in cultured muscle cells from patients with various deletions and point mutations, we now focus on single and multiple exon duplications. These mutations are in principle ideal targets for this approach since the specific skipping of duplicated exons would generate original, full-length transcripts.</p> <p>Methods</p> <p>Cultured muscle cells from DMD patients carrying duplications were transfected with AONs targeting the duplicated exons, and the dystrophin RNA and protein were analyzed.</p> <p>Results</p> <p>For two brothers with an exon 44 duplication, skipping was, even at suboptimal transfection conditions, so efficient that both exons 44 were skipped, thus generating, once more, an out-of-frame transcript. In such cases, one may resort to multi-exon skipping to restore the reading frame, as is shown here by inducing skipping of exon 43 and both exons 44. By contrast, in cells from a patient with an exon 45 duplication we were able to induce single exon 45 skipping, which allowed restoration of wild type dystrophin. The correction of a larger duplication (involving exons 52 to 62), by combinations of AONs targeting the outer exons, appeared problematic due to inefficient skipping and mistargeting of original instead of duplicated exons.</p> <p>Conclusion</p> <p>The correction of DMD duplications by exon skipping depends on the specific exons targeted. Its options vary from the ideal one, restoring for the first time the true, wild type dystrophin, to requiring more 'classical' skipping strategies, while the correction of multi-exon deletions may need the design of tailored approaches.</p

Exon Exchange Approach to Repair Duchenne Dystrophin Transcripts

Background: Trans-splicing strategies for mRNA repair involve engineered transcripts designed to anneal target mRNAs in order to interfere with their natural splicing, giving rise to mRNA chimeras where endogenous mutated exons have been replaced by exogenous replacement sequences. A number of trans-splicing molecules have already been proposed for replacing either the 59 or the 39 part of transcripts to be repaired. Here, we show the feasibility of RNA surgery by using a double trans-splicing approach allowing the specific substitution of a given mutated exon. Methodology/Principal Findings: As a target we used a minigene encoding a fragment of the mdx dystrophin gene enclosing the mutated exon (exon 23). This minigene was cotransfected with a variety of exon exchange constructions, differing in their annealing domains. We obtained accurate and efficient replacement of exon 23 in the mRNA target. Adding up a downstream intronic splice enhancer DISE in the exon exchange molecule enhanced drastically its efficiency up to 25–45 % of repair depending on the construction in use. Conclusions/Significance: These results demonstrate the possibility to fix up mutated exons, refurbish deleted exons and introduce protein motifs, while keeping natural untranslated sequences, which are essential for mRNA stability and translation regulation. Conversely to the well-known exon skipping, exon exchange has the advantage to be compatible with almost any type of mutations and more generally to a wide range of genetic conditions. In particular, it allows addressing disorders caused by dominant mutations

Multi-exon deletions of the FBN1 gene in Marfan syndrome

BACKGROUND: Mutations in the fibrillin -1 gene (FBN1) cause Marfan syndrome (MFS), an autosomal dominant multi-system connective tissue disorder. The 200 different mutations reported in the 235 kb, 65 exon-containing gene include only one family with a genomic multi-exon deletion. METHODS: We used long-range RT-PCR for mutation detection and long-range genomic PCR and DNA sequencing for identification of deletion breakpoints, allele-specific transcript analyses to determine stability of the mutant RNA, and pulse-chase studies to quantitate fibrillin synthesis and extracellular matrix deposition in cultured fibroblasts. Southern blots of genomic DNA were probed with three overlapping fragments covering the FBN1 coding exons RESULTS: Two novel multi-exon FBN1 deletions were discovered. Identical nucleotide pentamers were found at or near the intronic breakpoints. In a Case with classic MFS, an in-frame deletion of exons 42 and 43 removed the C-terminal 24 amino acids of the 5(th) LTBP (8-cysteine) domain and the adjacent 25(th) calcium-binding EGF-like (6-cysteine) domain. The mutant mRNA was stable, but fibrillin synthesis and matrix deposition were significantly reduced. A Case with severe childhood-onset MFS has a de novo deletion of exons 44–46 that removed three EGF-like domains. Fibrillin protein synthesis was normal, but matrix deposition was strikingly reduced. No genomic rearrangements were detected by Southern analysis of 18 unrelated MFS samples negative for FBN1 mutation screening. CONCLUSIONS: Two novel deletion cases expand knowledge of mutational mechanisms and genotype/phenotype correlations of fibrillinopathies. Deletions or mutations affecting an LTBP domain may result in unstable mutant protein cleavage products that interfere with microfibril assembly