Mohamed El-Kalioby

Unbiased targeted next-generation sequencing molecular approach for primary immunodeficiency diseases

BACKGROUND Molecular genetics techniques are an essential diagnostic tool for primary immunodeficiency diseases (PIDs). The use of next-generation sequencing (NGS) provides a comprehensive way of concurrently screening a large number of PID genes. However, its validity and cost-effectiveness require verification. OBJECTIVES We sought to identify and overcome complications associated with the use of NGS in a comprehensive gene panel incorporating 162 PID genes. We aimed to ascertain the specificity, sensitivity, and clinical sensitivity of the gene panel and its utility as a diagnostic tool for PIDs. METHODS A total of 162 PID genes were screened in 261 patients by using the Ion Torrent Proton NGS sequencing platform. Of the 261 patients, 122 had at least 1 known causal mutation at the onset of the study and were used to assess the specificity and sensitivity of the assay. The remaining samples were from unsolved cases that were biased toward more phenotypically and genotypically complicated cases. RESULTS The assay was able to detect the mutation in 117 (96%) of 122 positive control subjects with known causal mutations. For the unsolved cases, our assay resulted in a molecular genetic diagnosis for 35 of 139 patients. Interestingly, most of these cases represented atypical clinical presentations of known PIDs. CONCLUSIONS The targeted NGS PID gene panel is a sensitive and cost-effective diagnostic tool that can be used as a first-line molecular assay in patients with PIDs. The assay is an alternative choice to the complex and costly candidate gene approach, particularly for patients with atypical presentation of known PID genes.

Characterizing the morbid genome of ciliopathies

BackgroundCiliopathies are clinically diverse disorders of the primary cilium. Remarkable progress has been made in understanding the molecular basis of these genetically heterogeneous conditions; however, our knowledge of their morbid genome, pleiotropy, and variable expressivity remains incomplete.ResultsWe applied genomic approaches on a large patient cohort of 371 affected individuals from 265 families, with phenotypes that span the entire ciliopathy spectrum. Likely causal mutations in previously described ciliopathy genes were identified in 85% (225/265) of the families, adding 32 novel alleles. Consistent with a fully penetrant model for these genes, we found no significant difference in their “mutation load” beyond the causal variants between our ciliopathy cohort and a control non-ciliopathy cohort. Genomic analysis of our cohort further identified mutations in a novel morbid gene TXNDC15, encoding a thiol isomerase, based on independent loss of function mutations in individuals with a consistent ciliopathy phenotype (Meckel-Gruber syndrome) and a functional effect of its deficiency on ciliary signaling. Our study also highlighted seven novel candidate genes (TRAPPC3, EXOC3L2, FAM98C, C17orf61, LRRCC1, NEK4, and CELSR2) some of which have established links to ciliogenesis. Finally, we show that the morbid genome of ciliopathies encompasses many founder mutations, the combined carrier frequency of which accounts for a high disease burden in the study population.ConclusionsOur study increases our understanding of the morbid genome of ciliopathies. We also provide the strongest evidence, to date, in support of the classical Mendelian inheritance of Bardet-Biedl syndrome and other ciliopathies.

Clinical genomics can facilitate countrywide estimation of autosomal recessive disease burden

Background:Most autosomal recessive diseases are rare, but they collectively account for a substantial proportion of disease burden, especially in consanguineous populations. Estimation of this disease burden, however, is hampered by many factors, including lack of countrywide registries. Establishing carrier frequency can be a practical surrogate to estimate disease burden, although the requirement of a large representative cohort may be challenging.Purpose:We propose that the application of clinical genomics in the diagnostic setting offers a unique opportunity to estimate carrier frequency in the population as a secondary benefit.Methods:We used a data set of ~7,100 patients who underwent genomic testing for various Mendelian disorders to estimate the carrier frequency.Results:We were able to calculate the frequency of 259 confirmed founder recessive mutations. We found the corresponding disease burden to be, at minimum, ~7 per 1,000 children born to first-cousin parents, with disorders related to intellectual disability and vision impairment being the most common.Conclusion:Our approach can be utilized to inform the design of new policies for the prevention of genetic disorders and highlights an important secondary benefit of clinical genomics.Genet Med 18 12, 1244–1249.

Revisiting the morbid genome of Mendelian disorders

BackgroundThe pathogenicity of many Mendelian variants has been challenged by large-scale sequencing efforts. However, many rare and benign “disease mutations” are difficult to analyze due to their rarity. The Saudi Arabian variome is enriched for homozygosity due to inbreeding, a key advantage that can be exploited for the critical examination of previously published variants.ResultsWe collated all “disease-related mutations” listed in the Human Gene Mutation Database (HGMD) and ClinVar, including “variants of uncertain significance” (VOUS). We find that the use of public databases including 1000 Genomes, ExAC, and Kaviar can reclassify many of these variants as likely benign. Our Saudi Human Genome Program (SHGP) can reclassify many variants that are rare in public databases. Furthermore, SGPD allows us to observe many previously reported variants in the homozygous state and our extensive phenotyping of participants makes it possible to demonstrate the lack of phenotype for these variants, thus challenging their pathogenicity despite their rarity. We also find that 18 VOUS BRCA1 and BRCA2 variants that are listed in BRCA Exchange are present at least once in the homozygous state in patients who lack features of Fanconi anemia. Reassuringly, we could reciprocally demonstrate that none of those labeled as “pathogenic” were observed in the homozygous statue in individuals who lack Fanconi phenotype in our database.ConclusionOur study shows the importance of revisiting disease-related databases using public resources as well as of population-specific resources to improve the specificity of the morbid genome of Mendelian diseases in humans.

Streaming Support for Data Intensive Cloud-Based Sequence Analysis

Cloud computing provides a promising solution to the genomics data deluge problem resulting from the advent of next-generation sequencing (NGS) technology. Based on the concepts of “resources-on-demand” and “pay-as-you-go”, scientists with no or limited infrastructure can have access to scalable and cost-effective computational resources. However, the large size of NGS data causes a significant data transfer latency from the clients site to the cloud, which presents a bottleneck for using cloud computing services. In this paper, we provide a streaming-based scheme to overcome this problem, where the NGS data is processed while being transferred to the cloud. Our scheme targets the wide class of NGS data analysis tasks, where the NGS sequences can be processed independently from one another. We also provide the elastream package that supports the use of this scheme with individual analysis programs or with workflow systems. Experiments presented in this paper show that our solution mitigates the effect of data transfer latency and saves both time and cost of computation.

The Case for Docker in Multicloud Enabled Bioinformatics Applications

The introduction of next generation sequencing technologies did not bring only huge amounts of biological data but also highly sophisticated and versatile analysis workflows and systems. These new challenges require reliable and fast deployment methods over high performance servers in the local infrastructure or in the cloud. The use of virtualization technology has provided an efficient solution to overcome the complexity of deployment procedures and to provide a safe personalized execution box. However, the performance of applications running in virtual machines is worse than that of those running on the native infrastructure. Docker is a light weight alternative to the usual virtualization technology achieving notable better performance. In this paper, we explore the use case scenarios for using Docker to deploy and execute sophisticated bioinformatics tools and workflows, with a focus on the sequence analysis domain. We also introduce an efficient implementation of the package elasticHPC-Docker to enable creation of a docker-based computer cluster in the private cloud and in commercial clouds like Amazon and Google. We demonstrate by experiments that the use of elasticHPC-Docker is efficient and reliable in both private and commercial clouds.

Genetic spectrum of Saudi Arabian patients with antenatal cystic kidney disease and ciliopathy phenotypes using a targeted renal gene panel

Background Inherited cystic kidney disorders are a common cause of end-stage renal disease. Over 50 ciliopathy genes, which encode proteins that influence the structure and function of the primary cilia, are implicated in cystic kidney disease. Methods To define the phenotype and genotype of cystic kidney disease in fetuses and neonates, we correlated antenatal ultrasound examination and postnatal renal ultrasound examination with targeted exon sequencing, using a renal gene panel. A cohort of 44 families in whom antenatal renal ultrasound scanning findings in affected cases included bilateral cystic kidney disease, echogenic kidneys or enlarged kidneys was investigated. Results In this cohort, disease phenotypes were severe with 36 cases of stillbirth or perinatal death. Extra renal malformations, including encephalocele, polydactyly and heart malformations, consistent with ciliopathy phenotypes, were frequently detected. Renal gene panel testing identified causative mutations in 21 out of 34 families (62%), where patient and parental DNA was available. In the remaining 10 families, where only parental DNA was available, 7 inferred causative mutations were found. Together, mutations were found in 12 different genes with a total of 13 novel pathogenic variants, including an inferred novel variant in NEK8. Mutations in CC2D2A were the most common cause of an antenatal cystic kidney disease and a suspected ciliopathy in our cohort. Conclusions In families with ciliopathy phenotypes, mutational analysis using a targeted renal gene panel allows a rapid molecular diagnosis and provides important information for patients, parents and their physicians.

Supporting Bioinformatics Applications with Hybrid Multi-cloud Services

Cloud computing provides a promising solution to the big data problem associated with next generation sequencing applications. The increasing number of cloud service providers, who compete in terms of performance and price, is a clear indication of a growing market with high demand. However, current cloud computing based applications in bioinformatics do not profit from this progress, because they are still limited to just one cloud service provider. In this paper, we present different use case scenarios using hybrid services and resources from multiple cloud providers for bioinformatics applications. We also present a new version of the elasticHPC package to realize these scenarios and to support the creation of cloud computing resources over multiple cloud platforms, including Amazon, Google, Azure, and clouds supporting OpenStack. The instances created on these cloud environments are pre-configured to run big sequence analysis tasks using a large set of pre-installed software tools and parallelization techniques. In addition to its flexibility, we show by experiments that the use of hybrid cloud resources from different providers can save time and cost.

Genetic Profiling of Children with Advanced Cholestatic Liver Disease

Advanced cholestatic liver disease is a leading referral to pediatric liver transplant centers. Recent advances in the genetic classification of this group of disorders promise a highly personalized management although the genetic heterogeneity also poses a diagnostic challenge. Using a next‐generation sequencing‐based multi‐gene panel, we performed retrospective analysis of 98 pediatric patients who presented with advanced cholestatic liver disease. A likely causal mutation was identified in the majority (61%), spanning many genes including ones that have only rarely been reported to cause cholestatic liver disease, e.g. TJP2 and VIPAS39. We find no evidence to support mono‐allelic phenotypic expression in the carrier parents despite the severe nature of the respective mutations, and no evidence of oligogenicity. The high‐carrier frequency of the founder mutations identified in our cohort (1 in 87) suggests a minimum incidence of 1:7246, an alarmingly high disease burden that calls for the primary prevention through carrier screening.

WAMI: a web server for the analysis of minisatellite maps

BackgroundMinisatellites are genomic loci composed of tandem arrays of short repetitive DNA segments. A minisatellite map is a sequence of symbols that represents the tandem repeat array such that the set of symbols is in one-to-one correspondence with the set of distinct repeats. Due to variations in repeat type and organization as well as copy number, the minisatellite maps have been widely used in forensic and population studies. In either domain, researchers need to compare the set of maps to each other, to build phylogenetic trees, to spot structural variations, and to study duplication dynamics. Efficient algorithms for these tasks are required to carry them out reliably and in reasonable time.ResultsIn this paper we present WAMI, a web-server for the analysis of minisatellite maps. It performs the above mentioned computational tasks using efficient algorithms that take the model of map evolution into account. The WAMI interface is easy to use and the results of each analysis task are visualized.ConclusionsTo the best of our knowledge, WAMI is the first server providing all these computational facilities to the minisatellite community. The WAMI web-interface and the source code of the underlying programs are available at http://www.nubios.nileu.edu.eg/tools/wami.