Compound heterozygous mutations in RIPPLY2 associated with vertebral segmentation defects

Segmentation defects of the vertebrae (SDV) are caused by aberrant somite formation during embryogenesis and result in irregular formation of the vertebrae and ribs. The Notch signal transduction pathway plays a critical role in somite formation and patterning in model vertebrates. In humans, mutations in several genes involved in the Notch pathway are associated with SDV, with both autosomal recessive (MESP2, DLL3, LFNG, HES7) and autosomal dominant (TBX6) inheritance. However, many individuals with SDV do not carry mutations in these genes. Using whole-exome capture and massive parallel sequencing, we identified compound heterozygous mutations in RIPPLY2 in two brothers with multiple regional SDV, with appropriate familial segregation. One novel mutation (c.A238T:p.Arg80*) introduces a premature stop codon. In transiently transfected C2C12 mouse myoblasts, the RIPPLY2 mutant protein demonstrated impaired transcriptional repression activity compared with wild-type RIPPLY2 despite similar levels of expression. The other mutation (c.240-4T>G), with minor allele frequency <0.002, lies in the highly conserved splice site consensus sequence 5' to the terminal exon. Ripply2 has a well-established role in somitogenesis and vertebral column formation, interacting at both gene and protein levels with SDV-associated Mesp2 and Tbx6. We conclude that compound heterozygous mutations in RIPPLY2 are associated with SDV, a new gene for this condition. © The Author 2014.

Defects in the IFT-B component IFT172 cause Jeune and Mainzer-Saldino syndromes in humans

Intraflagellar transport (IFT) depends on two evolutionarily conserved modules, subcomplexes A (IFT-A) and B (IFT-B), to drive ciliary assembly and maintenance. All six IFT-A components and their motor protein, DYNC2H1, have been linked to human skeletal ciliopathies, including asphyxiating thoracic dystrophy (ATD; also known as Jeune syndrome), Sensenbrenner syndrome, and Mainzer-Saldino syndrome (MZSDS). Conversely, the 14 subunits in the IFT-B module, with the exception of IFT80, have unknown roles in human disease. To identify additional IFT-B components defective in ciliopathies, we independently performed different mutation analyses: candidate-based sequencing of all IFT-B-encoding genes in 1,467 individuals with a nephronophthisis-related ciliopathy or whole-exome resequencing in 63 individuals with ATD. We thereby detected biallelic mutations in the IFT-B-encoding gene IFT172 in 12 families. All affected individuals displayed abnormalities of the thorax and/or long bones, as well as renal, hepatic, or retinal involvement, consistent with the diagnosis of ATD or MZSDS. Additionally, cerebellar aplasia or hypoplasia characteristic of Joubert syndrome was present in 2 out of 12 families. Fibroblasts from affected individuals showed disturbed ciliary composition, suggesting alteration of ciliary transport and signaling. Knockdown of ift172 in zebrafish recapitulated the human phenotype and demonstrated a genetic interaction between ift172 and ift80. In summary, we have identified defects in IFT172 as a cause of complex ATD and MZSDS. Our findings link the group of skeletal ciliopathies to an additional IFT-B component, IFT172, similar to what has been shown for IFT-A.

Next generation sequencing identifies novel CACNA1A gene mutations in Episodic Ataxia type 2

Episodic Ataxia type 2 (EA2) is a rare autosomal dominantly inherited neurological disorder characterized by recurrent disabling imbalance, vertigo and episodes of ataxia lasting minutes to hours. EA2 is caused most often by loss of function mutations of the calcium channel gene CACNA1A. In addition to EA2, mutations in CACNA1A are responsible for two other allelic disorders: familial hemiplegic migraine type1 (FHM1) and spinocerebellar ataxia type 6 (SCA6). Herein, we have utilised Next Generation Sequencing (NGS) to screen the coding sequence, exon-intron boundaries and UTRs of five genes where mutation is known to produce symptoms related to EA2, including CACNA1A. We performed this screening in a group of 31 unrelated patients with EA2 symptoms. Both novel and known mutations were detected through NGS technology, and confirmed through Sanger sequencing. Genetic testing showed in total 15 mutation bearing patients (48%), of which 9 were novel mutations (6 missense and 3 small frameshift deletion mutations) and six known mutations (4 missense and 2 nonsense).These results demonstrate the efficiency of our NGS-panel for detecting known and novel mutations for EA2 in the CACNA1A gene, also identifying a novel missense mutation in ATP1A2 which is not a normal target for EA2 screening.

Ultra high throughput sequencing excludes MDH1 as candidate gene for RP28-linked retinitis pigmentosa

Purpose: Mutations in IDH3B, an enzyme participating in the Krebs cycle, have recently been found to cause autosomal recessive retinitis pigmentosa (arRP). The MDH1 gene maps within the RP28 arRP linkage interval and encodes cytoplasmic malate dehydrogenase, an enzyme functionally related to IDH3B. As a proof of concept for candidate gene screening to be routinely performed by ultra high throughput sequencing (UHTs), we analyzed MDH1 in a patient from each of the two families described so far to show linkage between arRP and RP28. Methods: With genomic long-range PCR, we amplified all introns and exons of the MDH1 gene (23.4 kb). PCR products were then sequenced by short-read UHTs with no further processing. Computer-based mapping of the reads and mutation detection were performed by three independent software packages. Results: Despite the intrinsic complexity of human genome sequences, reads were easily mapped and analyzed, and all algorithms used provided the same results. The two patients were homozygous for all DNA variants identified in the region, which confirms previous linkage and homozygosity mapping results, but had different haplotypes, indicating genetic or allelic heterogeneity. None of the DNA changes detected could be associated with the disease. Conclusions: The MDH1 gene is not the cause of RP28-linked arRP. Our experimental strategy shows that long-range genomic PCR followed by UHTs provides an excellent system to perform a thorough screening of candidate genes for hereditary retinal degeneration.

Sequencing of T-superfamily conotoxins from Conus virgo: Pyroglutamic acid identification and disulfide arrangement by MALDI mass spectrometry

De novo mass spectrometric sequencing of two Conus peptides, Vi1359 and Vi1361, from the vermivorous cone snail Conus virgo, found off the southern Indian coast, is presented. The peptides, whose masses differ only by 2 Da, possess two disulfide bonds and an amidated C-terminus. Simple chemical modifications and enzymatic cleavage coupled with matrix assisted laser desorption ionization (MALDI) mass spectrometric analysis aided in establishing the sequences of Vi1359, ZCCITIPECCRI-NH2, and Vi1361, ZCCPTMPECCRI-NH2, Which differ only at residues 4 and 6 (Z = pyroglutamic acid). The presence of the pyroglutamyl residue at the N-terminus was unambiguously identified by chemical hydrolysis of the cyclic amide, followed by esterification. The presence of Ile residues in both the peptides was confirmed from high-energy collision induced dissociation (CID) studies, using the observation Of W-n- and d(n)-ions as a diagnostic. Differential cysteine labeling, in conjunction with MALDI-MS/MS, permitted establishment of disulfide connectivity in both peptides as Cys2-Cys9 and Cys3-Cys10. The cysteine pattern clearly reveals that the peptides belong to the class of T-superfamily conotoxins, in particular the T-1 superfamily.

Ultra high throughput sequencing excludes MDH1 as candidate gene for RP28-linked retinitis pigmentosa

Purpose: Mutations in IDH3B, an enzyme participating in the Krebs cycle, have recently been found to cause autosomal recessive retinitis pigmentosa (arRP). The MDH1 gene maps within the RP28 arRP linkage interval and encodes cytoplasmic malate dehydrogenase, an enzyme functionally related to IDH3B. As a proof of concept for candidate gene screening to be routinely performed by ultra high throughput sequencing (UHTs), we analyzed MDH1 in a patient from each of the two families described so far to show linkage between arRP and RP28. Methods: With genomic long-range PCR, we amplified all introns and exons of the MDH1 gene (23.4 kb). PCR products were then sequenced by short-read UHTs with no further processing. Computer-based mapping of the reads and mutation detection were performed by three independent software packages. Results: Despite the intrinsic complexity of human genome sequences, reads were easily mapped and analyzed, and all algorithms used provided the same results. The two patients were homozygous for all DNA variants identified in the region, which confirms previous linkage and homozygosity mapping results, but had different haplotypes, indicating genetic or allelic heterogeneity. None of the DNA changes detected could be associated with the disease.

Optimization of sample preparation for next-generation sequencing with Illumina Genome Analyzer II

The growing interest for sequencing with higher throughput in the last decade has led to the development of new sequencing applications. This thesis concentrates on optimizing DNA library preparation for Illumina Genome Analyzer II sequencer. The library preparation steps that were optimized include fragmentation, PCR purification and quantification. DNA fragmentation was performed with focused sonication in different concentrations and durations. Two column based PCR purification method, gel matrix method and magnetic bead based method were compared. Quantitative PCR and gel electrophoresis in a chip were compared for DNA quantification. The magnetic bead purification was found to be the most efficient and flexible purification method. The fragmentation protocol was changed to produce longer fragments to be compatible with longer sequencing reads. Quantitative PCR correlates better with the cluster number and should thus be considered to be the default quantification method for sequencing. As a result of this study more data have been acquired from sequencing with lower costs and troubleshooting has become easier as qualification steps have been added to the protocol. New sequencing instruments and applications will create a demand for further optimizations in future.

Protein Model Discrimination Using Mutational Sensitivity Derived from Deep Sequencing

A major bottleneck in protein structure prediction is the selection of correct models from a pool of decoys. Relative activities of similar to 1,200 individual single-site mutants in a saturation library of the bacterial toxin CcdB were estimated by determining their relative populations using deep sequencing. This phenotypic information was used to define an empirical score for each residue (Rank Score), which correlated with the residue depth, and identify active-site residues. Using these correlations, similar to 98% of correct models of CcdB (RMSD <= 4 angstrom) were identified from a large set of decoys. The model-discrimination methodology was further validated on eleven different monomeric proteins using simulated RankScore values. The methodology is also a rapid, accurate way to obtain relative activities of each mutant in a large pool and derive sequence-structure-function relationships without protein isolation or characterization. It can be applied to any system in which mutational effects can be monitored by a phenotypic readout.

Multiple parents crossover operators: A new approach removes the overlapping solutions for sequencing problems

Maintaining population diversity throughout generations of Genetic Algorithms (GAs) is key to avoid premature convergence. Redundant solutions is one cause for the decreasing population diversity. To prevent the negative effect of redundant solutions, we propose a framework that is based on the multi-parents crossover (MPX) operator embedded in GAs. Because MPX generates diversified chromosomes with good solution quality, when a pair of redundant solutions is found, we would generate a new offspring by using the MPX to replace the redundant chromosome. Three schemes of MPX will be examined and will be compared against some algorithms in literature when we solve the permutation flowshop scheduling problems, which is a strong NP-Hard sequencing problem. The results indicate that our approach significantly improves the solution quality. This study is useful for researchers who are trying to avoid premature convergence of evolutionary algorithms by solving the sequencing problems.

Genome sequencing unveils a novel sea enterotoxin-carrying PVL phage in staphylococcus aureus ST772 from India

Staphylococcus aureus is a major human pathogen, first recognized as a leading cause of hospital-acquired infections. Community-associated S. aureus (CA-SA) pose a greater threat due to increase in severity of infection and disease among children and healthy adults. CA-SA strains in India are genetically diverse, among which is the sequence type (ST) 772, which has now spread to Australia, Europe and Japan. Towards understanding the genetic characteristics of ST772, we obtained draft genome sequences of five relevant clinical isolates and studied the properties of their PVL-carrying prophages, whose presence is a defining hallmark of CA-SA. We show that this is a novel prophage, which carries the structural genes of the hlb-carrying prophage and includes the sea enterotoxin. This architecture probably emerged early within the ST772 lineage, at least in India. The sea gene, unique to ST772 PVL, despite having promoter sequence characteristics typical of low expression, appears to be highly expressed during early phase of growth in laboratory conditions. We speculate that this might be a consequence of its novel sequence context. The crippled nature of the hlb-converting prophage in ST772. suggests that widespread mobility of the sea enterotoxin might be a selective force behind its `transfer' to the PVL prophage. Wild type ST772 strains induced strong proliferative responses as well as high cytotoxic activity against neutrophils, likely mediated by superantigen SEA and the PVL toxin respectively. Both proliferation and cytotoxicity were markedly reduced in a cured ST772 strain indicating the impact of the phage on virulence. The presence of SEA alongside he genes for the immune system-modulating PVL toxin may contribute to the success and virulence of ST772.

Residue specific contributions to stability and activity inferred from saturation mutagenesis and deep sequencing

Multiple methods currently exist for rapid construction and screening of single-site saturation mutagenesis (SSM) libraries in which every codon or nucleotide in a DNA fragment is individually randomized. Nucleotide sequences of each library member before and after screening or selection can be obtained through deep sequencing. The relative enrichment of each mutant at each position provides information on its contribution to protein activity or ligand-binding under the conditions of the screen. Such saturation scans have been applied to diverse proteins to delineate hot-spot residues, stability determinants, and for comprehensive fitness estimates. The data have been used to design proteins with enhanced stability, activity and altered specificity relative to wild-type, to test computational predictions of binding affinity, and for protein model discrimination. Future improvements in deep sequencing read lengths and accuracy should allow comprehensive studies of epistatic effects, of combinational variation at multiple sites, and identification of spatially proximate residues.

Identification of microRNAs and their targets in Finger millet by high throughput sequencing

MicroRNAs are short non-coding RNAs which play an important role in regulating gene expression by mRNA cleavage or by translational repression. The majority of identified miRNAs were evolutionarily conserved; however, others expressed in a species-specific manner. Finger millet is an important cereal crop; nonetheless, no practical information is available on microRNAs to date. In this study, we have identified 95 conserved microRNAs belonging to 39 families and 3 novel microRNAs by high throughput sequencing. For the identified conserved and novel miRNAs a total of 507 targets were predicted. 11 miRNAs were validated and tissue specificity was determined by stem loop RT-qPCR, Northern blot. GO analyses revealed targets of miRNA were involved in wide range of regulatory functions. This study implies large number of known and novel miRNAs found in Finger millet which may play important role in growth and development. (C) 2015 Elsevier B.V. All rights reserved.

Accurate sodium bisulfite sequencing in plants.

DNA cytosine methylation is a conserved epigenetic modification frequently correlating with transcriptional silencing in a wide variety of eukaryotic organisms. Sodium bisulfite treatment of DNA converts unmethylated cytosine to uracil, while 5-methylated cytosine is protected. We describe techniques that ensure reliable sequencing data following sodium bisulfite conversion and to avoid common pitfalls such as amplification of unconverted DNA and inclusion of sibling clones.

High-Throughput Sequencing of microRNAs in Peripheral Blood Mononuclear Cells: Identification of Potential Weight Loss Biomarkers

INTRODUCTION: MicroRNAs (miRNAs) are being increasingly studied in relation to energy metabolism and body composition homeostasis. Indeed, the quantitative analysis of miRNAs expression in different adiposity conditions may contribute to understand the intimate mechanisms participating in body weight control and to find new biomarkers with diagnostic or prognostic value in obesity management. OBJECTIVE: The aim of this study was the search for miRNAs in blood cells whose expression could be used as prognostic biomarkers of weight loss. METHODS: Ten Caucasian obese women were selected among the participants in a weight-loss trial that consisted in following an energy-restricted treatment. Weight loss was considered unsuccessful when <5% of initial body weight (non-responders) and successful when >5% (responders). At baseline, total miRNA isolated from peripheral blood mononuclear cells (PBMC) was sequenced with SOLiD v4. The miRNA sequencing data were validated by RT-PCR. RESULTS: Differential baseline expression of several miRNAs was found between responders and non-responders. Two miRNAs were up-regulated in the non-responder group (mir-935 and mir-4772) and three others were down-regulated (mir-223, mir-224 and mir-376b). Both mir-935 and mir-4772 showed relevant associations with the magnitude of weight loss, although the expression of other transcripts (mir-874, mir-199b, mir-766, mir-589 and mir-148b) also correlated with weight loss. CONCLUSIONS: This research addresses the use of high-throughput sequencing technologies in the search for miRNA expression biomarkers in obesity, by determining the miRNA transcriptome of PBMC. Basal expression of different miRNAs, particularly mir-935 and mir-4772, could be prognostic biomarkers and may forecast the response to a hypocaloric diet.