Identification Of Disease Genes Research Articles

Hunting human disease genes: lessons from the past, challenges for the future

The concept that a specific alteration in an individual’s DNA can result in disease is central to our notion of molecular medicine. The molecular basis of more than 3,500 Mendelian disorders has now been identified. In contrast, the identification of genes for common disease has been much more challenging. We discuss historical and contemporary approaches to disease gene identification, focusing on novel opportunities such as the use of population extremes and the identification of rare variants. While our ability to sequence DNA has advanced dramatically, assigning function to a given sequence change remains a major challenge, highlighting the need for both bioinformatics and functional approaches to appropriately interpret these data. We review progress in mapping and identifying human disease genes and discuss future challenges and opportunities for the field.

Mutations in phospholipase DDHD2 cause autosomal recessive hereditary spastic paraplegia (SPG54)

Hereditary spastic paraplegias (HSP) are a genetically heterogeneous group of disorders characterized by a distal axonopathy of the corticospinal tract motor neurons leading to progressive lower limb spasticity and weakness. Intracellular membrane trafficking, mitochondrial dysfunction and myelin formation are key functions involved in HSP pathogenesis. Only recently defects in metabolism of complex lipids have been implicated in a number of HSP subtypes. Mutations in the 23 known autosomal recessive HSP genes explain less than half of autosomal recessive HSP cases. To identify novel autosomal recessive HSP disease genes, exome sequencing was performed in 79 index cases with autosomal recessive forms of HSP. Resulting variants were filtered and intersected between families to allow identification of new disease genes. We identified two deleterious mutations in the phospholipase DDHD2 gene in two families with complicated HSP. The phenotype is characterized by early onset of spastic paraplegia, mental retardation, short stature and dysgenesis of the corpus callosum. Phospholipase DDHD2 is involved in intracellular membrane trafficking at the golgi/ endoplasmic reticulum interface and has been shown to possess phospholipase A1 activity in vitro. Discovery of DDHD2 mutations in HSP might therefore provide a link between two key pathogenic themes in HSP: membrane trafficking and lipid metabolism.

Dawn of ocular gene therapy: implications for molecular diagnosis in retinal disease

Personalized medicine aims to utilize genomic information about patients to tailor treatment. Gene replacement therapy for rare genetic disorders is perhaps the most extreme form of personalized medicine, in that the patients' genome wholly determines their treatment regimen. Gene therapy for retinal disorders is poised to become a clinical reality. The eye is an optimal site for gene therapy due to the relative ease of precise vector delivery, immune system isolation, and availability for monitoring of any potential damage or side effects. Due to these advantages, clinical trials for gene therapy of retinal diseases are currently underway. A necessary precursor to such gene therapies is accurate molecular diagnosis of the mutation(s) underlying disease. In this review, we discuss the application of Next Generation Sequencing (NGS) to obtain such a diagnosis and identify disease causing genes, using retinal disorders as a case study. After reviewing ocular gene therapy, we discuss the application of NGS to the identification of novel Mendelian disease genes. We then compare current, array based mutation detection methods against next NGS-based methods in three retinal diseases: Leber's Congenital Amaurosis, Retinitis Pigmentosa, and Stargardt's disease. We conclude that next-generation sequencing based diagnosis offers several advantages over array based methods, including a higher rate of successful diagnosis and the ability to more deeply and efficiently assay a broad spectrum of mutations. However, the relative difficulty of interpreting sequence results and the development of standardized, reliable bioinformatic tools remain outstanding concerns. In this review, recent advances NGS based molecular diagnoses are discussed, as well as their implications for the development of personalized medicine.

Telethon Network of Genetic Biobanks: a key service for diagnosis and research on rare diseases

Several examples have always illustrated how access to large numbers of biospecimens and associated data plays a pivotal role in the identification of disease genes and the development of pharmaceuticals. Hence, allowing researchers to access to significant numbers of quality samples and data, genetic biobanks are a powerful tool in basic, translational and clinical research into rare diseases. Recently demand for well-annotated and properly-preserved specimens is growing at a high rate, and is expected to grow for years to come. The best effective solution to this issue is to enhance the potentialities of well-managed biobanks by building a network.Here we report a 5-year experience of the Telethon Network of Genetic Biobanks (TNGB), a non-profit association of Italian repositories created in 2008 to form a virtually unique catalogue of biospecimens and associated data, which presently lists more than 750 rare genetic defects. The process of TNGB harmonisation has been mainly achieved through the adoption of a unique, centrally coordinated, IT infrastructure, which has enabled (i) standardisation of all the TNGB procedures and activities; (ii) creation of an updated TNGB online catalogue, based on minimal data set and controlled terminologies; (iii) sample access policy managed via a shared request control panel at web portal. TNGB has been engaged in disseminating information on its services into both scientific/biomedical - national and international - contexts, as well as associations of patients and families. Indeed, during the last 5-years national and international scientists extensively used the TNGB with different purposes resulting in more than 250 scientific publications. In addition, since its inception the TNGB is an associated member of the Biobanking and Biomolecular Resources Research Infrastructure and recently joined the EuroBioBank network. Moreover, the involvement of patients and families, leading to the formalization of various agreements between TNGB and Patients’ Associations, has demonstrated how promoting Biobank services can be instrumental in gaining a critical mass of samples essential for research, as well as, raising awareness, trust and interest of the general public in Biobanks. This article focuses on some fundamental aspects of networking and demonstrates how the translational research benefits from a sustained infrastructure.

Exome sequencing in roma families identifies tandem GRM1 mutations in a novel form of congenital cerebellar ataxia

Recessive ataxias are heterogeneous group of rare monogenic disorders with genetic pathology largely unresolved. Aim To identify the molecular defect underlying a novel congenital ataxia phenotype in a small Roma sub-isolate with a known strong founder effect. Methods The patients were assessed by an international multidis-ciplinary team. Whole exome sequencing data were used for inbreeding and relatedness estimates, linkage analysis and mutation detection. Functional mutation impact in patient lymphoblastoid cell lines was studied by reverse transcription/real-time PCR. Results The major clinical manifestations included global developmental delay, gait and stance ataxia, intellectual deficit, and progressive cerebellar atrophy. Novel tandem deletion c.2652_2654del:p.884_885del and a splicing mutation c.2660+2T>G, 7 bp apart in the metabotropic glutamate receptor gene GRM1, were the only homozygous variants in all affected subjects both exome-wide and within a unique linkage region. The splicing defect had a major functional impact, with exon skipping or transcript termination in the neighboring intron producing various species of nonfunctional receptors either lacking the trans-membrane domain and containing abnormal intracellular tails or completely missing the tail. Discussion The study is the first to implicate GRM1 in hereditary ataxia and to highlight the potential of exome sequencing in the Roma population for disease gene and mutation identification

A Primer for Disease Gene Prioritization Using Next-Generation Sequencing Data

High-throughput next-generation sequencing (NGS) technology produces a tremendous amount of raw sequence data. The challenges for researchers are to process the raw data, to map the sequences to genome, to discover variants that are different from the reference genome, and to prioritize/rank the variants for the question of interest. The recent development of many computational algorithms and programs has vastly improved the ability to translate sequence data into valuable information for disease gene identification. However, the NGS data analysis is complex and could be overwhelming for researchers who are not familiar with the process. Here, we outline the analysis pipeline and describe some of the most commonly used principles and tools for analyzing NGS data for disease gene identification.

A vertex similarity-based framework to discover and rank orphan disease-related genes

BackgroundA rare or orphan disease (OD) is any disease that affects a small percentage of the population. While opportunities now exist to accelerate progress toward understanding the basis for many more ODs, the prioritization of candidate genes is still a critical step for disease-gene identification. Several network-based frameworks have been developed to address this problem with varied results.ResultWe have developed a novel vertex similarity (VS) based parameter-free prioritizing framework to identify and rank orphan disease candidate genes. We validate our approach by using 1598 known orphan disease-causing genes (ODGs) representing 172 orphan diseases (ODs). We compare our approach with a state-of-art parameter-based approach (PageRank with Priors or PRP) and with another parameter-free method (Interconnectedness or ICN). Our results show that VS-based approach outperforms ICN and is comparable to PRP. We further apply VS-based ranking to identify and rank potential novel candidate genes for several ODs.ConclusionWe demonstrate that VS-based parameter-free ranking approach can be successfully used for disease candidate gene prioritization and can complement other network-based methods for candidate disease gene ranking. Importantly, our VS-ranked top candidate genes for the ODs match the known literature, suggesting several novel causal relationships for further investigation.

Asphyxiating Thoracic Dysplasia: clinical and molecular review of 42 families

Asphyxiating Thoracic Dysplasia (ATD) ((MIM 208500, MIM 6112633, MIM 613091, MIM 61819, MIM614376) belongs to the short rib polydactyly group and is characterized by a long and narrow thorax, short long bones and trident acetabular roof. Polydactyly, retinal degeneration, cystic renal and liver diseases have been occasionally reported. Today, mutations in IFT80 (MIM 611177), DYNC2H1 (MIM 603297), TCC21B (MIM 612014) and WDR19 (MIM 608151) genes have been reported in ATD. Through a national grant (PHRC, AOM 06031), we have collected 55 ATD cases including 29 fetuses issued from 42 families who benefit the combined approach of deep phenotyping and molecular screening of IFT80 and DYNC2H1. The series included 26 alive cases ranging in age from 6 months to 36 years. Respiratory treatment was needed in 46%, including positive pression respiration, and invasive or non-invasive ventilation. Cystic renal and liver diseases occur in 16% of cases; whereas retinal degeneration was present in 40 % cases aged more than 2 years (6/15). The molecular screening allowed us to detect DYNC2H1 mutations in 63% and IFT80 mutations in 6%. In 6 cases, only one heterozygote mutation in either IFT80 or DYNC2H1 was identified. Finally, the two genes were excluded in 31% cases. These preliminary results emphasize that DYNC2H1 is the major gene responsible for ATD. The presence of only one mutation (27% of mutated cases) may suggest a digenic diallelic inheritance. Ongoing studies will hopefully lead to the identification of other disease genes.

The Muscular Dystrophies

The muscular dystrophies are disorders of progressive muscular degeneration and weakness. As a group they display clinical heterogeneity that reflects the heterogeneity of molecular mechanisms responsible for them, and range from congenital to adulthood onset. Recent advances in the field include improved methods of diagnosis, continued identification of disease genes, and the development of a unified model of pathogenesis in facioscapulohumeral dystrophy. These advances are reflected in the development of new therapeutic approaches, some of which have already led to clinical trials in the dystrophinopathies and limb-girdle dystrophies.

A novel homozygous mutation in SUCLA2 gene identified by exome sequencing

Mitochondrial disorders with multiple mitochondrial respiratory chain (MRC) enzyme deficiency and depletion of mitochondrial DNA (mtDNA) are autosomal recessive conditions due to mutations in several nuclear genes necessary for proper mtDNA maintenance.In this report, we describe two Italian siblings presenting with encephalomyopathy and mtDNA depletion in muscle. By whole exome-sequencing and prioritization of candidate genes, we identified a novel homozygous missense mutation in the SUCLA2 gene in a highly conserved aminoacid residue. Although a recurrent mutation in the SUCLA2 gene is relatively frequent in the Faroe Islands, mutations in other populations are extremely rare. In contrast with what has been reported in other patients, methyl-malonic aciduria, a biomarker for this genetic defect, was absent in our proband and very mildly elevated in her affected sister.This report demonstrates that next-generation technologies, particularly exome-sequencing, are user friendly, powerful means for the identification of disease genes in genetically and clinically heterogeneous inherited conditions, such as mitochondrial disorders.

A novel nonsense CDK5RAP2 mutation in a Somali child with primary microcephaly and sensorineural hearing loss

Primary microcephaly is a genetically heterogeneous condition characterized by reduced head circumference (−3 SDS or more) and mild-to-moderate learning disability. Here, we describe clinical and molecular investigations of a microcephalic child with sensorineural hearing loss. Although consanguinity was unreported initially, detection of 13.7 Mb of copy neutral loss of heterozygosity (cnLOH) on chromosome 9 implicated the CDK5RAP2 gene. Targeted sequencing identified a homozygous E234X mutation, only the third mutation to be described in CDK5RAP2, the first in an individual of non-Pakistani descent. Sensorineural hearing loss is not generally considered to be consistent with autosomal recessive microcephaly and therefore it seems likely that the deafness in this individual is caused by the co-occurrence of a further gene mutation, independent of CDK5RAP2. Nevertheless, further detailed clinical descriptions of rare CDK5RAP2 patients, including hearing assessments will be needed to resolve fully the phenotypic range associated with mutations in this gene. This study also highlights the utility of SNP-array testing to guide disease gene identification where an autosomal recessive condition is plausible. © 2012 Wiley Periodicals, Inc.

A generalizable pre-clinical research approach for orphan disease therapy

With the advent of next-generation DNA sequencing, the pace of inherited orphan disease gene identification has increased dramatically, a situation that will continue for at least the next several years. At present, the numbers of such identified disease genes significantly outstrips the number of laboratories available to investigate a given disorder, an asymmetry that will only increase over time. The hope for any genetic disorder is, where possible and in addition to accurate diagnostic test formulation, the development of therapeutic approaches. To this end, we propose here the development of a strategic toolbox and preclinical research pathway for inherited orphan disease. Taking much of what has been learned from rare genetic disease research over the past two decades, we propose generalizable methods utilizing transcriptomic, system-wide chemical biology datasets combined with chemical informatics and, where possible, repurposing of FDA approved drugs for pre-clinical orphan disease therapies. It is hoped that this approach may be of utility for the broader orphan disease research community and provide funding organizations and patient advocacy groups with suggestions for the optimal path forward. In addition to enabling academic pre-clinical research, strategies such as this may also aid in seeding startup companies, as well as further engaging the pharmaceutical industry in the treatment of rare genetic disease.

The sound and the fly:Drosophilain biomedical research on human hearing

Deep biochemical and cellular similarities exist between Drosophila and vertebrates in the development and function of all organs. Despite their morphological differences, it is considered ever more likely that the hearing sensory cells have a common evolutionary origin and that consequently the molecular mechanisms that govern their functions are at least partially conserved. Thanks to transgenesis techniques and to the rich collection of mutant stocks well characterized from genetic, molecular and physiological points of view, Drosophila provides the means to test in vivo vertebrate gene functions in known metabolic and cellular pathways. In addition, the search for sequence homologies between human and Drosophila genes has allowed the identification of many disease genes in the human auditory system. These results indicate that Drosophila is a good model for the study of the genes that cause deafness in humans, and offers the possibility of using the numerous and sophisticated genetic tools of the inse...

Gene identification in the congenital disorders of glycosylation type I by whole-exome sequencing

Congenital disorders of glycosylation type I (CDG-I) form a growing group of recessive neurometabolic diseases. Identification of disease genes is compromised by the enormous heterogeneity in clinical symptoms and the large number of potential genes involved. Until now, gene identification included the sequential application of biochemical methods in blood samples and fibroblasts. In genetically unsolved cases, homozygosity mapping has been applied in consanguineous families. Altogether, this time-consuming diagnostic strategy led to the identification of defects in 17 different CDG-I genes. Here, we applied whole-exome sequencing (WES) in combination with the knowledge of the protein N-glycosylation pathway for gene identification in our remaining group of six unsolved CDG-I patients from unrelated non-consanguineous families. Exome variants were prioritized based on a list of 76 potential CDG-I candidate genes, leading to the rapid identification of one known and two novel CDG-I gene defects. These included the first X-linked CDG-I due to a de novo mutation in ALG13, and compound heterozygous mutations in DPAGT1, together the first two steps in dolichol-PP-glycan assembly, and mutations in PGM1 in two cases, involved in nucleotide sugar biosynthesis. The pathogenicity of the mutations was confirmed by showing the deficient activity of the corresponding enzymes in patient fibroblasts. Combined with these results, the gene defect has been identified in 98% of our CDG-I patients. Our results implicate the potential of WES to unravel disease genes in the CDG-I in newly diagnosed singleton families.

Identifying disease genes and module biomarkers by differential interactions

A complex disease is generally caused by the mutation of multiple genes or by the dysfunction of multiple biological processes. Systematic identification of causal disease genes and module biomarkers can provide insights into the mechanisms underlying complex diseases, and help develop efficient therapies or effective drugs. In this paper, we present a novel approach to predict disease genes and identify dysfunctional networks or modules, based on the analysis of differential interactions between disease and control samples, in contrast to the analysis of differential gene or protein expressions widely adopted in existing methods. As an example, we applied our method to the study of three-stage microarray data for gastric cancer. We identified network modules or module biomarkers that include a set of genes related to gastric cancer, implying the predictive power of our method. The results on holdout validation data sets show that our identified module can serve as an effective module biomarker for accurately detecting or diagnosing gastric cancer, thereby validating the efficiency of our method. We proposed a new approach to detect module biomarkers for diseases, and the results on gastric cancer demonstrated that the differential interactions are useful to detect dysfunctional modules in the molecular interaction network, which in turn can be used as robust module biomarkers.

Congenital diaphragmatic hernia candidate genes derived from embryonic transcriptomes

Congenital diaphragmatic hernia (CDH) is a common (1 in 3,000 live births) major congenital malformation that results in significant morbidity and mortality. The discovery of CDH loci using standard genetic approaches has been hindered by its genetic heterogeneity. We hypothesized that gene expression profiling of developing embryonic diaphragms would help identify genes likely to be associated with diaphragm defects. We generated a time series of whole-transcriptome expression profiles from laser captured embryonic mouse diaphragms at embryonic day (E)11.5 and E12.5 when experimental perturbations lead to CDH phenotypes, and E16.5 when the diaphragm is fully formed. Gene sets defining biologically relevant pathways and temporal expression trends were identified by using a series of bioinformatic algorithms. These developmental sets were then compared with a manually curated list of genes previously shown to cause diaphragm defects in humans and in mouse models. Our integrative filtering strategy identified 27 candidates for CDH. We examined the diaphragms of knockout mice for one of the candidate genes, pre-B-cell leukemia transcription factor 1 (Pbx1), and identified a range of previously undetected diaphragmatic defects. Our study demonstrates the utility of genetic characterization of normal development as an integral part of a disease gene identification and prioritization strategy for CDH, an approach that can be extended to other diseases and developmental anomalies.

Computational approaches to disease‐gene prediction: rationale, classification and successes

The identification of genes involved in human hereditary diseases often requires the time-consuming and expensive examination of a great number of possible candidate genes, since genome-wide techniques such as linkage analysis and association studies frequently select many hundreds of 'positional' candidates. Even considering the positive impact of next-generation sequencing technologies, the prioritization of candidate genes may be an important step for disease-gene identification. In this paper we develop a basic classification scheme for computational approaches to disease-gene prediction and apply it to exhaustively review bioinformatics tools that have been developed for this purpose, focusing on conceptual aspects rather than technical detail and performance. Finally, we discuss some past successes obtained by computational approaches to illustrate their beneficial contribution to medical research.

Mutations in kelch-like 3 and cullin 3 cause hypertension and electrolyte abnormalities

Hypertension affects one billion people and is a principal reversible risk factor for cardiovascular disease. A rare Mendelian syndrome, pseudohypoaldosteronism type II (PHAII), featuring hypertension, hyperkalemia, and metabolic acidosis, has revealed previously unrecognized physiology orchestrating the balance between renal salt reabsorption versus K+ and H+ excretion1. We used exome sequencing to identify mutations in Kelch-like 3 (KLHL3) or Cullin 3 (CUL3) in 41 PHAII kindreds. KLHL3 mutations are either recessive or dominant, while CUL3 mutations are dominant and predominantly de novo. CUL3 and BTB-Kelch proteins such as KLHL3 are components of Cullin/RING E3 ligase complexes (CRLs) that ubiquitinate substrates bound to Kelch propeller domains2–8. Dominant KLHL3 mutations are clustered in short segments within the Kelch propeller and BTB domains implicated in substrate9 and Cullin5 binding, respectively. Diverse CUL3 mutations all result in skipping of exon 9, producing an in-frame deletion. Because dominant KLHL3 and CUL3 mutations both phenocopy recessive loss-of-function KLHL3 mutations, they may abrogate ubiquitination of KLHL3 substrates. Disease features are reversed by thiazide diuretics, which inhibit the Na-Cl cotransporter (NCC) in the distal nephron of the kidney; KLHL3 and CUL3 are expressed in this location, suggesting a mechanistic link between KLHL3/CUL3 mutations, increased Na-Cl reabsorption, and disease pathogenesis. These findings demonstrate the utility of exome sequencing in disease gene identification despite combined complexities of locus heterogeneity, mixed models of transmission, and frequent de novo mutation, and establish a fundamental role for KLHL3/CUL3 in blood pressure, K+, and pH homeostasis.

Disease gene identification strategies for exome sequencing.

Next generation sequencing can be used to search for Mendelian disease genes in an unbiased manner by sequencing the entire protein-coding sequence, known as the exome, or even the entire human genome. Identifying the pathogenic mutation amongst thousands to millions of genomic variants is a major challenge, and novel variant prioritization strategies are required. The choice of these strategies depends on the availability of well-phenotyped patients and family members, the mode of inheritance, the severity of the disease and its population frequency. In this review, we discuss the current strategies for Mendelian disease gene identification by exome resequencing. We conclude that exome strategies are successful and identify new Mendelian disease genes in approximately 60% of the projects. Improvements in bioinformatics as well as in sequencing technology will likely increase the success rate even further. Exome sequencing is likely to become the most commonly used tool for Mendelian disease gene identification for the coming years.

Genetic Mapping and Exome Sequencing Identify Variants Associated with Five Novel Diseases

The Clinic for Special Children (CSC) has integrated biochemical and molecular methods into a rural pediatric practice serving Old Order Amish and Mennonite (Plain) children. Among the Plain people, we have used single nucleotide polymorphism (SNP) microarrays to genetically map recessive disorders to large autozygous haplotype blocks (mean = 4.4 Mb) that contain many genes (mean = 79). For some, uninformative mapping or large gene lists preclude disease-gene identification by Sanger sequencing. Seven such conditions were selected for exome sequencing at the Broad Institute; all had been previously mapped at the CSC using low density SNP microarrays coupled with autozygosity and linkage analyses. Using between 1 and 5 patient samples per disorder, we identified sequence variants in the known disease-causing genes SLC6A3 and FLVCR1, and present evidence to strongly support the pathogenicity of variants identified in TUBGCP6, BRAT1, SNIP1, CRADD, and HARS. Our results reveal the power of coupling new genotyping technologies to population-specific genetic knowledge and robust clinical data.