The growing availability of human genetic variation has given rise to novel methods of measuring genetic tolerance that better interpret variants of unknown significance. We recently developed a ...concept based on protein domain homology in the human genome to improve variant interpretation. For this purpose, we mapped population variation from the Exome Aggregation Consortium (ExAC) and pathogenic mutations from the Human Gene Mutation Database (HGMD) onto Pfam protein domains. The aggregation of these variation data across homologous domains into meta‐domains allowed us to generate amino acid resolution of genetic intolerance profiles for human protein domains. Here, we developed MetaDome, a fast and easy‐to‐use web server that visualizes meta‐domain information and gene‐wide profiles of genetic tolerance. We updated the underlying data of MetaDome to contain information from 56,319 human transcripts, 71,419 protein domains, 12,164,292 genetic variants from gnomAD, and 34,076 pathogenic mutations from ClinVar. MetaDome allows researchers to easily investigate their variants of interest for the presence or absence of variation at corresponding positions within homologous domains. We illustrate the added value of MetaDome by an example that highlights how it may help in the interpretation of variants of unknown significance. The MetaDome web server is freely accessible at https://stuart.radboudumc.nl/metadome.
We developed MetaDome, a fast and easy‐to‐use web server that visualizes meta‐domain information and gene‐wide profiles of genetic tolerance. MetaDome allows researchers to easily investigate their variants of interest for the presence or absence of variation at corresponding positions within homologous domains. The MetaDome web server is freely accessible at https://stuart.radboudumc.nl/metadome.
The genetic underpinnings of the most common adult-onset neurodegenerative disorders (AOND) are complex in majority of the cases. In some families, however, the disease can be inherited in a ...Mendelian fashion as an autosomal-dominant trait. Next to that, patients carrying mutations in the same disease genes have been reported despite a negative family history. Although challenging to demonstrate due to the late onset of the disease in most cases, the occurrence of de novo mutations can explain this sporadic presentation, as demonstrated for severe neurodevelopmental disorders. Exome or genome sequencing of patient–parent trios allows a hypothesis-free study of the role of de novo mutations in AOND and the discovery of novel disease genes. Another hypothesis that may explain a proportion of sporadic AOND cases is the occurrence of a de novo mutation after the fertilization of the oocyte (post-zygotic mutation) or even as a late-somatic mutation, restricted to the brain. Such somatic mutation hypothesis, that can be tested with the use of novel sequencing technologies, is fully compatible with the seeding and spreading mechanisms of the pathological proteins identified in most of these disorders. We review here the current knowledge and future perspectives on de novo mutations in known and novel candidate genes identified in the most common AONDs such as Alzheimer’s disease, Parkinson’s disease, the frontotemporal lobar degeneration spectrum and Prion disorders. Also, we review the first lessons learned from recent genomic studies of control and diseased brains and the challenges which remain to be addressed.
De novo mutations are recognized both as an important source of genetic variation and as a prominent cause of sporadic disease in humans. Mutations identified as de novo are generally assumed to have ...occurred during gametogenesis and, consequently, to be present as germline events in an individual. Because Sanger sequencing does not provide the sensitivity to reliably distinguish somatic from germline mutations, the proportion of de novo mutations that occur somatically rather than in the germline remains largely unknown. To determine the contribution of post-zygotic events to de novo mutations, we analyzed a set of 107 de novo mutations in 50 parent-offspring trios. Using four different sequencing techniques, we found that 7 (6.5%) of these presumed germline de novo mutations were in fact present as mosaic mutations in the blood of the offspring and were therefore likely to have occurred post-zygotically. Furthermore, genome-wide analysis of “de novo” variants in the proband led to the identification of 4/4,081 variants that were also detectable in the blood of one of the parents, implying parental mosaicism as the origin of these variants. Thus, our results show that an important fraction of de novo mutations presumed to be germline in fact occurred either post-zygotically in the offspring or were inherited as a consequence of low-level mosaicism in one of the parents.
ABSTRACT
For next‐generation sequencing technologies, sufficient base‐pair coverage is the foremost requirement for the reliable detection of genomic variants. We investigated whether whole‐genome ...sequencing (WGS) platforms offer improved coverage of coding regions compared with whole‐exome sequencing (WES) platforms, and compared single‐base coverage for a large set of exome and genome samples. We find that WES platforms have improved considerably in the last years, but at comparable sequencing depth, WGS outperforms WES in terms of covered coding regions. At higher sequencing depth (95x–160x), WES successfully captures 95% of the coding regions with a minimal coverage of 20x, compared with 98% for WGS at 87‐fold coverage. Three different assessments of sequence coverage bias showed consistent biases for WES but not for WGS. We found no clear differences for the technologies concerning their ability to achieve complete coverage of 2,759 clinically relevant genes. We show that WES performs comparable to WGS in terms of covered bases if sequenced at two to three times higher coverage. This does, however, go at the cost of substantially more sequencing biases in WES approaches. Our findings will guide laboratories to make an informed decision on which sequencing platform and coverage to choose.
For next‐generation sequencing technologies sufficient base‐pair coverage is the foremost requirement for the reliable detection of genomic variants. We investigated whether whole genome sequencing (WGS) platforms offer improved coverage of coding regions compared to whole exome sequencing (WES) platforms. We show that WES performs comparable to WGS in terms of covered bases if sequenced at 2–3 times higher coverage. This does, however go at the cost of substantially more sequencing biases in WES approaches.
Osteogenesis imperfecta (OI) is a heterogeneous genetic disorder characterized by bone fragility and susceptibility to fractures after minimal trauma. After mutations in all known OI genes had been ...excluded by Sanger sequencing, we applied next-generation sequencing to analyze the exome of a single individual who has a severe form of the disease and whose parents are second cousins. A total of 26,922 variations from the human reference genome sequence were subjected to several filtering steps. In addition, we extracted the genotypes of all dbSNP130-annotated SNPs from the exome sequencing data and used these 299,494 genotypes as markers for the genome-wide identification of homozygous regions. A single homozygous truncating mutation, affecting
SERPINF1 on chromosome 17p13.3, that was embedded into a homozygous stretch of 2.99 Mb remained. The mutation was also homozygous in the affected brother of the index patient. Subsequently, we identified homozygosity for two different truncating
SERPINF1 mutations in two unrelated patients with OI and parental consanguinity. All four individuals with
SERPINF1 mutations have severe OI. Fractures of long bones and severe vertebral compression fractures with resulting deformities were observed as early as the first year of life in these individuals. Collagen analyses with cultured dermal fibroblasts displayed no evidence for impaired collagen folding, posttranslational modification, or secretion.
SERPINF1 encodes pigment epithelium-derived factor (PEDF), a secreted glycoprotein of the serpin superfamily. PEDF is a multifunctional protein and one of the strongest inhibitors of angiogenesis currently known in humans. Our data provide genetic evidence for PEDF involvement in human bone homeostasis.
To identify candidate genes for intellectual disability, we performed a meta-analysis on 2,637 de novo mutations, identified from the exomes of 2,104 patient-parent trios. Statistical analyses ...identified 10 new candidate ID genes: DLG4, PPM1D, RAC1, SMAD6, SON, SOX5, SYNCRIP, TCF20, TLK2 and TRIP12. In addition, we show that these genes are intolerant to nonsynonymous variation and that mutations in these genes are associated with specific clinical ID phenotypes.
De novo mutations (DNMs) originating in gametogenesis are an important source of genetic variation. We use a data set of 7,216 autosomal DNMs with resolved parent of origin from whole-genome ...sequencing of 816 parent-offspring trios to investigate differences between maternally and paternally derived DNMs and study the underlying mutational mechanisms. Our results show that the number of DNMs in offspring increases not only with paternal age, but also with maternal age, and that some genome regions show enrichment for maternally derived DNMs. We identify parent-of-origin-specific mutation signatures that become more pronounced with increased parental age, pointing to different mutational mechanisms in spermatogenesis and oogenesis. Moreover, we find DNMs that are spatially clustered to have a unique mutational signature with no significant differences between parental alleles, suggesting a different mutational mechanism. Our findings provide insights into the molecular mechanisms that underlie mutagenesis and are relevant to disease and evolution in humans.
Haploinsufficiency (HI) is the best characterized mechanism through which dominant mutations exert their effect and cause disease. Non-haploinsufficiency (NHI) mechanisms, such as gain-of-function ...and dominant-negative mechanisms, are often characterized by the spatial clustering of mutations, thereby affecting only particular regions or base pairs of a gene. Variants leading to haploinsufficency might occasionally cluster as well, for example in critical domains, but such clustering is on the whole less pronounced with mutations often spread throughout the gene. Here we exploit this property and develop a method to specifically identify genes with significant spatial clustering patterns of de novo mutations in large cohorts. We apply our method to a dataset of 4,061 de novo missense mutations from published exome studies of trios with intellectual disability and developmental disorders (ID/DD) and successfully identify 15 genes with clustering mutations, including 12 genes for which mutations are known to cause neurodevelopmental disorders. For 11 out of these 12, NHI mutation mechanisms have been reported. Additionally, we identify three candidate ID/DD-associated genes of which two have an established role in neuronal processes. We further observe a higher intolerance to normal genetic variation of the identified genes compared to known genes for which mutations lead to HI. Finally, 3D modeling of these mutations on their protein structures shows that 81% of the observed mutations are unlikely to affect the overall structural integrity and that they therefore most likely act through a mechanism other than HI.
Implementation of novel genetic diagnostic tests is generally driven by technological advances because they promise shorter turnaround times and/or higher diagnostic yields. Other aspects, including ...impact on clinical management or cost-effectiveness, are often not assessed in detail prior to implementation.
We studied the clinical utility of whole-exome sequencing (WES) in complex pediatric neurology in terms of diagnostic yield and costs. We analyzed 150 patients (and their parents) presenting with complex neurological disorders of suspected genetic origin. In a parallel study, all patients received both the standard diagnostic workup (e.g., cerebral imaging, muscle biopsies or lumbar punctures, and sequential gene-by-gene-based testing) and WES simultaneously.
Our unique study design allowed direct comparison of diagnostic yield of both trajectories and provided insight into the economic implications of implementing WES in this diagnostic trajectory. We showed that WES identified significantly more conclusive diagnoses (29.3%) than the standard care pathway (7.3%) without incurring higher costs. Exploratory analysis of WES as a first-tier diagnostic test indicates that WES may even be cost-saving, depending on the extent of other tests being omitted.
Our data support such a use of WES in pediatric neurology for disorders of presumed genetic origin.Genet Med advance online publication 23 March 2017.
The substantial technological advancements in next-generation sequencing (NGS), combined with dropping costs, have allowed for a swift diffusion of NGS applications in clinical settings. Although ...several commercial parties report to have broken the $1000 barrier for sequencing an entire human genome, a valid cost overview for NGS is currently lacking. This study provides a complete, transparent and up-to-date overview of the total costs of different NGS applications.
Cost calculations for targeted gene panels (TGP), whole exome sequencing (WES) and whole genome sequencing (WGS) were based on the Illumina NextSeq500, HiSeq4000, and HiSeqX5 platforms, respectively. To anticipate future developments, sensitivity analyses are performed.
Per-sample costs were €1669 for WGS, € 792 for WES and €333 for TGP. To reach the coveted $1000 genome, not only is the long-term and efficient use of the sequencing equipment needed, but also large reductions in capital costs and especially consumable costs are also required.
WES and TGP are considerably lower-cost alternatives to WGS. However, this does not imply that these NGS approaches should be preferred in clinical practice, since this should be based on the tradeoff between costs and the expected clinical utility of the approach chosen. The results of the present study contribute to the evaluation of such tradeoffs.
PDF
