Genome sequencing data analysis for rare disease gene discovery.

Rare diseases occur in a smaller proportion of the general population, which is variedly defined as less than 200 000 individuals (US) or in less than 1 in 2000 individuals (Europe). Although rare, they collectively make up to approximately 7000 different disorders, with majority having a genetic origin, and affect roughly 300 million people globally. Most of the patients and their families undergo a long and frustrating diagnostic odyssey. However, advances in the field of genomics have started to facilitate the process of diagnosis, though it is hindered by the difficulty in genome data analysis and interpretation. A major impediment in diagnosis is in the understanding of the diverse approaches, tools and datasets available for variant prioritization, the most important step in the analysis of millions of variants to select a few potential variants. Here we present a review of the latest methodological developments and spectrum of tools available for rare disease genetic variant discovery and recommend appropriate data interpretation methods for variant prioritization. We have categorized the resources based on various steps of the variant interpretation workflow, starting from data processing, variant calling, annotation, filtration and finally prioritization, with a special emphasis on the last two steps. The methods discussed here pertain to elucidating the genetic basis of disease in individual patient cases via trio- or family-based analysis of the genome data. We advocate the use of a combination of tools and datasets and to follow multiple iterative approaches to elucidate the potential causative variant.

Annexin A3 in sepsis: novel perspectives from an exploration of public transcriptome data.

According to publicly available transcriptome datasets, the abundance of Annexin A3 (ANXA3) is robustly increased during the course of sepsis; however, no studies have examined the biological significance or clinical relevance of ANXA3 in this pathology. Here we explored this interpretation gap and identified possible directions for future research. Based on reference transcriptome datasets, we found that ANXA3 expression is restricted to neutrophils, is upregulated in vitro after exposure to plasma obtained from septic patients, and is associated with adverse clinical outcomes. Secondly, an increase in ANXA3 transcript abundance was also observed in vivo, in the blood of septic patients in multiple independent studies. ANXA3 is known to mediate calcium-dependent granules-phagosome fusion in support of microbicidal activity in neutrophils. More recent work has also shown that ANXA3 enhances proliferation and survival of tumour cells via a Caspase-3-dependent mechanism. And this same molecule is also known to play a critical role in regulation of apoptotic events in neutrophils. Thus, we posit that during sepsis ANXA3 might either play a beneficial role, by facilitating microbial clearance and resolution of the infection; or a detrimental role, by prolonging neutrophil survival, which is known to contribute to sepsis-mediated organ damage.

The Arabidopsis RNA-binding protein AtRGGA regulates tolerance to salt and drought stress.

Salt and drought stress severely reduce plant growth and crop productivity worldwide. The identification of genes underlying stress response and tolerance is the subject of intense research in plant biology. Through microarray analyses, we previously identified in potato (Solanum tuberosum) StRGGA, coding for an Arginine Glycine Glycine (RGG) box-containing RNA-binding protein, whose expression was specifically induced in potato cell cultures gradually exposed to osmotic stress. Here, we show that the Arabidopsis (Arabidopsis thaliana) ortholog, AtRGGA, is a functional RNA-binding protein required for a proper response to osmotic stress. AtRGGA gene expression was up-regulated in seedlings after long-term exposure to abscisic acid (ABA) and polyethylene glycol, while treatments with NaCl resulted in AtRGGA down-regulation. AtRGGA promoter analysis showed activity in several tissues, including stomata, the organs controlling transpiration. Fusion of AtRGGA with yellow fluorescent protein indicated that AtRGGA is localized in the cytoplasm and the cytoplasmic perinuclear region. In addition, the rgga knockout mutant was hypersensitive to ABA in root growth and survival tests and to salt stress during germination and at the vegetative stage. AtRGGA-overexpressing plants showed higher tolerance to ABA and salt stress on plates and in soil, accumulating lower levels of proline when exposed to drought stress. Finally, a global analysis of gene expression revealed extensive alterations in the transcriptome under salt stress, including several genes such as ASCORBATE PEROXIDASE2, GLUTATHIONE S-TRANSFERASE TAU9, and several SMALL AUXIN UPREGULATED RNA-like genes showing opposite expression behavior in transgenic and knockout plants. Taken together, our results reveal an important role of AtRGGA in the mechanisms of plant response and adaptation to stress.

Increased Relative Abundance of Ruminoccocus Is Associated With Reduced Cardiovascular Risk in an Obese Population.

Background: Obesity is a complex disease with underlying genetic, environmental, psychological, physiological, medical, and epigenetic factors. Obesity can cause various disorders, including cardiovascular diseases (CVDs), that are among the most prevalent chronic conditions in Qatar. Recent studies have highlighted the significant roles of the gut microbiome in improving the pathology of various diseases, including obesity. Thus, in this study, we aimed to investigate the effects of dietary intake and gut microbial composition in modulating the risk of CVD development in obese Qatari adults. Methods: We enrolled 46 adult subjects (18-65 years of age) who were classified based on their CVD risk scores, calculated using the Framingham formula, into a CVD no-risk group (score of <10%, n = 36) and CVD risk group (score of ≥10%, n = 10). For each study subject, we measured the gut microbial composition with a 16s rDNA sequencing method that targeted the v3-v4 region using Illumina Miseq, and their nutritional status was recorded based on 24-h dietary recall. Dietary intake, bacterial taxa summary, diversity index, microbial markers, pathway analysis, and network correlation were determined for the study subjects. Results: The CVD risk group showed a lower intake of vitamin D, reduced relative abundance of genera Ruminococcus and Bifidobacterium, no change in bacterial diversity, and higher levels of taurine, hypotaurine, and lipoic acid metabolism than the CVD no-risk group. Besides, the relative abundance of genus Ruminococcus was positively correlated with the intake of protein, monounsaturated fat, vitamin A, and vitamin D. Conclusion: Taken together, our results suggest that the genus Ruminococcus could be used as a microbial marker, and its reduced relative abundance could mediate the risk of CVDs in the Obese Qatari population.

Can the Salivary Microbiome Predict Cardiovascular Diseases? Lessons Learned From the Qatari Population.

Background: Many studies have linked dysbiosis of the gut microbiome to the development of cardiovascular diseases (CVD). However, studies assessing the association between the salivary microbiome and CVD risk on a large cohort remain sparse. This study aims to identify whether a predictive salivary microbiome signature is associated with a high risk of developing CVD in the Qatari population. Methods: Saliva samples from 2,974 Qatar Genome Project (QGP) participants were collected from Qatar Biobank (QBB). Based on the CVD score, subjects were classified into low-risk (LR < 10) (n = 2491), moderate-risk (MR = 10-20) (n = 320) and high-risk (HR > 30) (n = 163). To assess the salivary microbiome (SM) composition, 16S-rDNA libraries were sequenced and analyzed using QIIME-pipeline. Machine Learning (ML) strategies were used to identify SM-based predictors of CVD risk. Results: Firmicutes and Bacteroidetes were the predominant phyla among all the subjects included. Linear Discriminant Analysis Effect Size (LEfSe) analysis revealed that Clostridiaceae and Capnocytophaga were the most significantly abundant genera in the LR group, while Lactobacillus and Rothia were significantly abundant in the HR group. ML based prediction models revealed that Desulfobulbus, Prevotella, and Tissierellaceae were the common predictors of increased risk to CVD. Conclusion: This study identified significant differences in the SM composition in HR and LR CVD subjects. This is the first study to apply ML-based prediction modeling using the SM to predict CVD in an Arab population. More studies are required to better understand the mechanisms of how those microbes contribute to CVD.

Akkermansia, a Possible Microbial Marker for Poor Glycemic Control in Qataris Children Consuming Arabic Diet-A Pilot Study on Pediatric T1DM in Qatar.

In Qatar, Type 1 Diabetes mellitus (T1DM) is one of the most prevalent disorders. This study aimed to explore the gut microbiome's relation to the continuous subcutaneous insulin infusion (CSII) therapy, dietary habits, and the HbA1c level in the pediatric T1DM subjects in Qatar. We recruited 28 T1DM subjects with an average age of 10.5 ± 3.53 years. The stool sample was used to measure microbial composition by 16s rDNA sequencing method. The results have revealed that the subjects who had undergone CSII therapy had increased microbial diversity and genus Akkermansia was significantly enriched in the subjects without CSII therapy. Moreover, genus Akkermansia was higher in the subjects with poor glycemic control (HbA1c > 7.5%). When we classified the subjects based on dietary patterns and nationality, Akkermansia was significantly enriched in Qataris subjects without the CSII therapy consuming Arabic diet than expatriates living in Qatar and eating a Western/mixed diet. Thus, this pilot study showed that abundance of Akkermansia is dependent on the Arabic diet only in poorly controlled Qataris T1DM patients, opening new routes to personalized treatment for T1DM in Qataris pediatric subjects. Further comprehensive studies on the relation between the Arabic diet, ethnicity, and Akkermansia are warranted to confirm this preliminary finding.

Bifidobacterium reduction is associated with high blood pressure in children with type 1 diabetes mellitus.

Children with Type 1 diabetes mellitus (T1DM) have an elevated risk of abnormal blood pressure (BP) measurements and patterns. Both hypertension and T1DM are well-known risk factors for cardiovascular disease and kidney failure. The human microbiome has been linked to both diabetes and hypertension, but the relationship between the gut microbiome and BP in children with T1DM is not well-understood. In this cross-sectional study, we examined the relationship between resting office BP and gut microbiota composition, diversity, and richness in children with T1DM and healthy controls. We recruited 29 pediatric subjects and divided them into three groups: healthy controls (HC, n = 5), T1DM with normal BP (T1DM-Normo, n = 17), and T1DM with elevated BP (T1DM-HBP, n = 7). We measured the BP, dietary and clinical parameters for each subject. We collected fecal samples to perform the 16s rDNA sequencing and to measure the short-chain fatty acids (SCFAs) level. The microbiome downstream analysis included the relative abundance of microbiota, alpha and beta diversity, microbial markers using Linear Discriminant effect size analysis (LEfSe), potential gut microbial metabolic pathways using Phylogenetic Investigation of Communities by Reconstruction of Unobserved States (PICRUSt) and metabolic pathways validation using Statistical Inference of Associations between Microbial Communities And host phenotype (SIAMCAT) machine learning toolbox. Our study results showed that T1DM-HBP group had distinct gut microbial composition (at multiple taxonomic levels) and reduced diversity (richness and abundance) compared with T1DM-Normo and HC groups. Children with T1DM-HBP showed a significant reduction of Bifidobacterium levels (especially B. adolescentis, B. bifidum, and B. longum) compared to the T1DM-Normo group. We also observed unique gut-microbial metabolic pathways, such as elevated lipopolysaccharide synthesis and glutathione metabolism in children with T1DM-HBP compared to T1DM-Normo children. We can conclude that the reduction in the abundance of genus Bifidobacterium could play a significant role in elevating the BP in pediatric T1DM subjects. More studies are needed to corroborate our findings and further explore the potential contributing mechanisms we describe.

Human and mouse oligonucleotide-based array CGH.

Array-based comparative genomic hybridization is a high resolution method for measuring chromosomal copy number changes. Here we present a validated protocol using in-house spotted oligonucleotide libraries for array comparative genomic hybridization (CGH). This oligo array CGH platform yields reproducible results and is capable of detecting single copy gains, multi-copy amplifications as well as homozygous and heterozygous deletions as small as 100 kb with high resolution. A human oligonucleotide library was printed on amine binding slides. Arrays were hybridized using a hybstation and analysed using BlueFuse feature extraction software, with >95% of spots passing quality control. The protocol allows as little as 300 ng of input DNA and a 90% reduction of Cot-1 DNA without compromising quality. High quality results have also been obtained with DNA from archival tissue. Finally, in addition to human oligo arrays, we have applied the protocol successfully to mouse oligo arrays. We believe that this oligo-based platform using 'off-the-shelf' oligo libraries provides an easy accessible alternative to BAC arrays for CGH, which is cost-effective, available at high resolution and easily implemented for any sequenced organism without compromising the quality of the results.

THY1 is a candidate tumour suppressor gene with decreased expression in metastatic nasopharyngeal carcinoma.

Using oligonucleotide microarray analysis, THY1, mapping close to a previously defined 11q22-23 nasopharyngeal carcinoma (NPC) critical region was identified as showing consistent downregulated expression in the tumour segregants, as compared to their parental tumour-suppressing microcell hybrids (MCHs). Gene expression and protein analyses show that THY1 was not expressed in the NPC HONE1 recipient cells, tumour segregants, and other NPC cell lines; THY1 was exclusively expressed in the non-tumourigenic MCHs. The mechanism of THY1 gene inactivation in these cell lines was attributed to hypermethylation. Clinical study showed that in 65% of NPC specimens there was either downregulation or loss of THY1 gene expression. Using a tissue microarray and immunohistochemical staining, 44% of the NPC cases showed downregulated expression of THY1 and 9% lost THY1 expression. The frequency of THY1 downregulated expression in lymph node metastatic NPC was 63%, which was significantly higher than in the primary tumour (33%). After transfection of THY1 gene into HONE1 cells, a dramatic reduction of colony formation ability was observed. These findings suggest that THY1 is a good candidate tumour suppressor gene in NPC, which is significantly associated with lymph node metastases.

BEARR: Batch Extraction and Analysis of cis-Regulatory Regions.

Transcription factors play important roles in regulating biological and disease processes. Microarray technology has enabled researchers to simultaneously monitor changes in the expression of thousands of transcripts. By identifying specific transcription factor binding sites in the cis-regulatory regions of differentially expressed genes, it is then possible to identify direct targets of transcription factors, model transcriptional regulatory networks and mine the dataset for relevant targets for experimental and clinical manipulation. We have developed web-based software to assist biologists in efficiently carrying out the analysis of microarray data from studies of specific transcription factors. Batch Extraction and Analysis of cis-Regulatory Regions, or BEARR, accepts gene identifier lists from microarray data analysis tools and facilitates identification, extraction and analysis of regulatory regions from the large amount of data that is typically generated in these types of studies. The software is publicly available at http://giscompute.gis.a-star.edu.sg/~vega/BEARR1.0/.

Functional analysis of a cell cycle-associated, tumor-suppressive gene, protein tyrosine phosphatase receptor type G, in nasopharyngeal carcinoma.

Functional studies to identify the potential role of a chromosome 3p14-21 gene, protein tyrosine phosphatase receptor type G (PTPRG), were performed. PTPRG was identified as a candidate tumor suppressor gene (TSG) in nasopharyngeal carcinoma (NPC) by differential gene profiling of tumorigenic and nontumorigenic NPC chromosome 3 microcell hybrids (MCH). Down-regulation of this gene was found in tumor segregants when compared with their corresponding tumor-suppressive MCHs, as well as in NPC cell lines and tumor biopsies. Promoter hypermethylation and loss of heterozygosity were found to be important mechanisms contributing to PTPRG silencing. PTPRG overexpression in NPC cell lines induces growth suppression and reduced anchorage-independent growth in vitro. This is the first study to use a tetracycline-responsive vector expression system to study PTPRG stable transfectants. Results indicate its ability to induce significant tumor growth suppression in nude mice under conditions activating transgene expression. These studies now provide functional evidence indicating critical interactions of PTPRG in the extracellular matrix milieu induce cell arrest and changes in cell cycle status. This is associated with inhibition of pRB phosphorylation through down-regulation of cyclin D1. These novel findings enhance our current understanding of how PTPRG may contribute to tumorigenesis.