Challenges in Whole Exome Sequencing: An Example from Hereditary Deafness

Whole exome sequencing provides unprecedented opportunities to identify causative DNA variants in rare Mendelian disorders. Finding the responsible mutation via traditional methods in families with hearing loss is difficult due to a high degree of genetic heterogeneity. In this study we combined autozygosity mapping and whole exome sequencing in a family with 3 affected children having nonsyndromic hearing loss born to consanguineous parents. Two novel missense homozygous variants, c.508C>A (p.H170N) in GIPC3 and c.1328C>T (p.T443M) in ZNF57, were identified in the same ∼6 Mb autozygous region on chromosome 19 in affected members of the family. Both variants co-segregated with the phenotype and were absent in 335 ethnicity-matched controls. Biallelic GIPC3 mutations have recently been reported to cause autosomal recessive nonsyndromic sensorineural hearing loss. Thus we conclude that the hearing loss in the family described in this report is caused by a novel missense mutation in GIPC3. Identified variant in GIPC3 had a low read depth, which was initially filtered out during the analysis leaving ZNF57 as the only potential causative gene. This study highlights some of the challenges in the analyses of whole exome data in the bid to establish the true causative variant in Mendelian disease

The serine protease domain of MASP-3: enzymatic properties and crystal structure in complex with ecotin.

International audienceMannan-binding lectin (MBL), ficolins and collectin-11 are known to associate with three homologous modular proteases, the MBL-Associated Serine Proteases (MASPs). The crystal structures of the catalytic domains of MASP-1 and MASP-2 have been solved, but the structure of the corresponding domain of MASP-3 remains unknown. A link between mutations in the MASP1/3 gene and the rare autosomal recessive 3MC (Mingarelli, Malpuech, Michels and Carnevale,) syndrome, characterized by various developmental disorders, was discovered recently, revealing an unexpected important role of MASP-3 in early developmental processes. To gain a first insight into the enzymatic and structural properties of MASP-3, a recombinant form of its serine protease (SP) domain was produced and characterized. The amidolytic activity of this domain on fluorescent peptidyl-aminomethylcoumarin substrates was shown to be considerably lower than that of other members of the C1r/C1s/MASP family. The E. coli protease inhibitor ecotin bound to the SP domains of MASP-3 and MASP-2, whereas no significant interaction was detected with MASP-1, C1r and C1s. A tetrameric complex comprising an ecotin dimer and two MASP-3 SP domains was isolated and its crystal structure was solved and refined to 3.2 Å. Analysis of the ecotin/MASP-3 interfaces allows a better understanding of the differential reactivity of the C1r/C1s/MASP protease family members towards ecotin, and comparison of the MASP-3 SP domain structure with those of other trypsin-like proteases yields novel hypotheses accounting for its zymogen-like properties in vitro

Biochemical evaluation of oxidative stress during exercise in patients with coronary heart disease

The impact of exercise tolerance test on oxidative stress was assessed by thiobarbituric acid reactive substances and markers of antioxidant status, namely Cu Zn superoxide dismutase, glutathione peroxidase, glutathione and vitamin E in blood samples of patients with exertional angina. The study was aimed to differentiate patients with positive exercise test (coronary heart disease patients) from patients with negative exercise test, at rest and peak exercise with respect to the investigated variables. Significantly lower values for both glutathione peroxidase activity and glutathione level were observed in patients after exercise test (p<0.01 and p<0.05, respectively). Only the patients with positive exercise test had significantly lower values for Cu Zn superoxide dismutase, glutathione peroxidase and glutathione, and a significantly higher ratio of thiobarbituric acid reactive substances/glutathione after exercise, as compared to before (p<0.05, p<0.05, p<0.05, p<0.01, respectively). Our findings indicate that the exercise test applied to patients with exertional angina oxidatively stresses the erythrocytes to a greater extent in exercise test (+) patients than in exercise test (-) patients

Plasmonic Metasurfaces Situated on Ultrathin Carbon Nanomembranes

During the past decade, optical metasurfaces consisting of designed nanoresonators arranged in a planar fashion were successfully demonstrated to allow for the realization of a large variety of flat optical components. However, in common implementations of metasurfaces and metasurface-based devices, their flat nature is thwarted by the presence of a substrate of macroscopic thickness, which is needed to mechanically support the individual nanoresonators. Here, we demonstrate that carbon nanomembranes (CNMs) having nanoscale thicknesses can be used as a basis for arranging an array of plasmonic nanoresonators into a metamembrane, allowing for the realization of genuinely flat optical devices. CNMs belong to the family of two-dimensional materials, and their thicknesses and mechanical, chemical, and electrical properties can be tailored by the choice of the molecular precursors used for their fabrication. We experimentally fabricate gold split-ring-resonator (SRR) metasurfaces on top of a free-standing CNM, which has a thickness of only about 1 nm and shows a negligible interaction with the incident light field. For optical characterization of the fabricated SRR CNM metasurfaces, we perform linear-optical transmittance spectroscopy, revealing the typical resonance structure of an SRR metasurface. Furthermore, numerical calculations assuming free-standing SRR arrays are in good overall agreement with corresponding experimental transmittance spectra. We believe that our scheme offers a versatile solution for the realization of ultrathin, ultra lightweight metadevices, and may initiate various future research directions and applications including complex sensor technologies, conformal coating of complex topographies with functional metasurfaces, fast prototyping of multilayer metasurfaces, and studying the optical properties of effectively free-standing nanoparticles without the need for levitation schemes

GATAD2B loss-of-function mutations cause a recognisable syndrome with intellectual disability and are associated with learning deficits and synaptic undergrowth in Drosophila

Contains fulltext : 124865.pdf (publisher's version ) (Open Access)BACKGROUND: GATA zinc finger domain containing 2B (GATAD2B) encodes a subunit of the MeCP1-Mi-2/nucleosome remodelling and deacetylase complex involved in chromatin modification and regulation of transcription. We recently identified two de novo loss-of-function mutations in GATAD2B by whole exome sequencing in two unrelated individuals with severe intellectual disability. METHODS: To identify additional individuals with GATAD2B aberrations, we searched for microdeletions overlapping with GATAD2B in inhouse and international databases, and performed targeted Sanger sequencing of the GATAD2B locus in a selected cohort of 80 individuals based on an overlap with the clinical features in the two index cases. To address whether GATAD2B is required directly in neurones for cognition and neuronal development, we investigated the role of Drosophila GATAD2B orthologue simjang (simj) in learning and synaptic connectivity. RESULTS: We identified a third individual with a 240 kb microdeletion encompassing GATAD2B and a fourth unrelated individual with GATAD2B loss-of-function mutation. Detailed clinical description showed that all four individuals with a GATAD2B aberration had a distinctive phenotype with childhood hypotonia, severe intellectual disability, limited speech, tubular shaped nose with broad nasal tip, short philtrum, sparse hair and strabismus. Neuronal knockdown of Drosophila GATAD2B orthologue, simj, resulted in impaired learning and altered synapse morphology. CONCLUSIONS: We hereby define a novel clinically recognisable intellectual disability syndrome caused by loss-of-function of GATAD2B. Our results in Drosophila suggest that GATAD2B is required directly in neurones for normal cognitive performance and synapse development

A canonical splice site mutation in GIPC3 causes sensorineural hearing loss in a large Pakistani family

Item does not contain fulltextWith homozygosity mapping we have identified two large homozygous regions on chromosome 3q13.11-q13.31 and chromosome 19p13.3-q31.32 in a large Pakistani family suffering from autosomal recessive nonsyndromic hearing impairment (arNSHI). The region on chromosome 19 overlaps with the previously described deafness loci DFNB15, DFNB72 and DFNB95. Mutations in GIPC3 have been shown to underlie the nonsyndromic hearing impairment linked to these loci. Sequence analysis of all exons and exon-intron boundaries of GIPC3 revealed a homozygous canonical splice site mutation, c.226-1G>T, in GIPC3. This is the first mutation described in GIPC3 that affects splicing. The c.226-1G>T mutation is located in the acceptor splice site of intron 1 and is predicted to affect the normal splicing of exon 2. With a minigene assay it was shown to result in the use of an alternative acceptor site in exon 2, resulting in a frameshift and a premature stop codon. This study expands the mutational spectrum of GIPC3 in arNSHI