Induced pluripotent stem cells (iPSCs) are an established cellular system to study the impact of genetic variants in derived cell types and developmental contexts. However, in their pluripotent ...state, the disease impact of genetic variants is less well known. Here, we integrate data from 1,367 human iPSC lines to comprehensively map common and rare regulatory variants in human pluripotent cells. Using this population-scale resource, we report hundreds of new colocalization events for human traits specific to iPSCs, and find increased power to identify rare regulatory variants compared with somatic tissues. Finally, we demonstrate how iPSCs enable the identification of causal genes for rare diseases.
The ClinGen Inborn Errors of Metabolism Working Group was tasked with creating a comprehensive, standardized knowledge base of genes and variants for metabolic diseases. Phenylalanine hydroxylase ...(PAH) deficiency was chosen to pilot development of the Working Group's standards and guidelines. A PAH variant curation expert panel (VCEP) was created to facilitate this process. Following ACMG‐AMP variant interpretation guidelines, we present the development of these standards in the context of PAH variant curation and interpretation. Existing ACMG‐AMP rules were adjusted based on disease (6) or strength (5) or both (2). Disease adjustments include allele frequency thresholds, functional assay thresholds, and phenotype‐specific guidelines. Our validation of PAH‐specific variant interpretation guidelines is presented using 85 variants. The PAH VCEP interpretations were concordant with existing interpretations in ClinVar for 69 variants (81%). Development of biocurator tools and standards are also described. Using the PAH‐specific ACMG‐AMP guidelines, 714 PAH variants have been curated and will be submitted to ClinVar. We also discuss strategies and challenges in applying ACMG‐AMP guidelines to autosomal recessive metabolic disease, and the curation of variants in these genes.
The ClinGen Inborn Errors of Metabolism Working Group chose phenylalanine hydroxylase (PAH) deficiency to pilot metabolic‐specific ACMG‐AMP variant interpretation guidelines. A PAH variant curation expert panel adjusted existing ACMG‐AMP rules based on disease (6) or strength (5) or both (2). Disease adjustments include allele frequency thresholds, functional assay thresholds, and phenotype‐specific guidelines. Our validation of PAH‐specific variant interpretation guidelines are presented. Using these guidelines 714 PAH variants have been curated and will be submitted to ClinVar.
Biallelic variants in IL6ST, encoding GP130, cause a recessive form of hyper-IgE syndrome (HIES) characterized by high IgE level, eosinophilia, defective acute phase response, susceptibility to ...bacterial infections, and skeletal abnormalities due to cytokine-selective loss of function in GP130, with defective IL-6 and IL-11 and variable oncostatin M (OSM) and IL-27 levels but sparing leukemia inhibitory factor (LIF) signaling.
Our aim was to understand the functional and structural impact of recessive HIES-associated IL6ST variants.
We investigated a patient with HIES by using exome, genome, and RNA sequencing. Functional assays assessed IL-6, IL-11, IL-27, OSM, LIF, CT-1, CLC, and CNTF signaling. Molecular dynamics simulations and structural modeling of GP130 cytokine receptor complexes were performed.
We identified a patient with compound heterozygous novel missense variants in IL6ST (p.Ala517Pro and the exon-skipping null variant p.Gly484_Pro518delinsArg). The p.Ala517Pro variant resulted in a more profound IL-6– and IL-11–dominated signaling defect than did the previously identified recessive HIES IL6ST variants p.Asn404Tyr and p.Pro498Leu. Molecular dynamics simulations suggested that the p.Ala517Pro and p.Asn404Tyr variants result in increased flexibility of the extracellular membrane–proximal domains of GP130. We propose a structural model that explains the cytokine selectivity of pathogenic IL6ST variants that result in recessive HIES. The variants destabilized the conformation of the hexameric cytokine receptor complexes, whereas the trimeric LIF-GP130-LIFR complex remained stable through an additional membrane-proximal interaction. Deletion of this membrane-proximal interaction site in GP130 consequently caused additional defective LIF signaling and Stüve-Wiedemann syndrome.
Our data provide a structural basis to understand clinical phenotypes in patients with IL6ST variants.
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KCTD7 is a member of the potassium channel tetramerization domain-containing protein family and has been associated with progressive myoclonic epilepsy (PME), characterized by myoclonus, epilepsy, ...and neurological deterioration. Here we report four affected individuals from two unrelated families in which we identified KCTD7 compound heterozygous single nucleotide variants through exome sequencing. RNAseq was used to detect a non-annotated splicing junction created by a synonymous variant in the second family. Whole-cell patch-clamp analysis of neuroblastoma cells overexpressing the patients' variant alleles demonstrated aberrant potassium regulation. While all four patients experienced many of the common clinical features of PME, they also showed variable phenotypes not previously reported, including dysautonomia, brain pathology findings including a significantly reduced thalamus, and the lack of myoclonic seizures. To gain further insight into the pathogenesis of the disorder, zinc finger nucleases were used to generate kctd7 knockout zebrafish. Kctd7 homozygous mutants showed global dysregulation of gene expression and increased transcription of c-fos, which has previously been correlated with seizure activity in animal models. Together these findings expand the known phenotypic spectrum of KCTD7-associated PME, report a new animal model for future studies, and contribute valuable insights into the disease.
Celotno besedilo
Dostopno za:
DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
Identification of clinically significant genetic alterations involved in human disease has been dramatically accelerated by developments in next-generation sequencing technologies. However, the ...infrastructure and accessible comprehensive curation tools necessary for analyzing an individual patient genome and interpreting genetic variants to inform healthcare management have been lacking.
Here we present the ClinGen Variant Curation Interface (VCI), a global open-source variant classification platform for supporting the application of evidence criteria and classification of variants based on the ACMG/AMP variant classification guidelines. The VCI is among a suite of tools developed by the NIH-funded Clinical Genome Resource (ClinGen) Consortium and supports an FDA-recognized human variant curation process. Essential to this is the ability to enable collaboration and peer review across ClinGen Expert Panels supporting users in comprehensively identifying, annotating, and sharing relevant evidence while making variant pathogenicity assertions. To facilitate evidence-based improvements in human variant classification, the VCI is publicly available to the genomics community. Navigation workflows support users providing guidance to comprehensively apply the ACMG/AMP evidence criteria and document provenance for asserting variant classifications.
The VCI offers a central platform for clinical variant classification that fills a gap in the learning healthcare system, facilitates widespread adoption of standards for clinical curation, and is available at https://curation.clinicalgenome.org.
Decreased sequencing costs have led to an explosion of genetic and genomic data. These data have revealed thousands of candidate human disease variants. Establishing which variants cause phenotypes ...and diseases, however, has remained challenging. Significant progress has been made, including advances by the National Institutes of Health (NIH)-funded Undiagnosed Diseases Network (UDN). However, 6000-13,000 additional disease genes remain to be identified. The continued discovery of rare diseases and their genetic underpinnings provides benefits to affected patients, of whom there are more than 400 million worldwide, and also advances understanding the mechanisms of more common diseases. Platforms employing model organisms enable discovery of novel gene-disease relationships, help establish variant pathogenicity, and often lead to the exploration of underlying mechanisms of pathophysiology that suggest new therapies. The Model Organism Screening Center (MOSC) of the UDN is a unique resource dedicated to utilizing informatics and functional studies in model organisms, including worm (Caenorhabditis elegans), fly (Drosophila melanogaster), and zebrafish (Danio rerio), to aid in diagnosis. The MOSC has directly contributed to the diagnosis of challenging cases, including multiple patients with complex, multi-organ phenotypes. In addition, the MOSC provides a framework for how basic scientists and clinicians can collaborate to drive diagnoses. Customized experimental plans take into account patient presentations, specific genes and variant(s), and appropriateness of each model organism for analysis. The MOSC also generates bioinformatic and experimental tools and reagents for the wider scientific community. Two elements of the MOSC that have been instrumental in its success are (1) multidisciplinary teams with expertise in variant bioinformatics and in human and model organism genetics, and (2) mechanisms for ongoing communication with clinical teams. Here we provide a position statement regarding the central role of model organisms for continued discovery of disease genes, and we advocate for the continuation and expansion of MOSC-type research entities as a Model Organisms Network (MON) to be funded through grant applications submitted to the NIH, family groups focused on specific rare diseases, other philanthropic organizations, industry partnerships, and other sources of support.
Sixty to seventy-five percent of individuals with rare and undiagnosed phenotypes remain undiagnosed after exome sequencing (ES). With standard ES reanalysis resolving 10–15% of the ES negatives, ...further approaches are necessary to maximize diagnoses in these individuals.
In 38 ES negative patients an individualized genomic–phenotypic approach was employed utilizing (1) phenotyping; (2) reanalyses of FASTQ files, with innovative bioinformatics; (3) targeted molecular testing; (4) genome sequencing (GS); and (5) conferring of clinical diagnoses when pathognomonic clinical findings occurred.
Certain and highly likely diagnoses were made in 18/38 (47%) individuals, including identifying two new developmental disorders. The majority of diagnoses (>70%) were due to our bioinformatics, phenotyping, and targeted testing identifying variants that were undetected or not prioritized on prior ES. GS diagnosed 3/18 individuals with structural variants not amenable to ES. Additionally, tentative diagnoses were made in 3 (8%), and in 5 individuals (13%) candidate genes were identified. Overall, diagnoses/potential leads were identified in 26/38 (68%).
Our comprehensive approach to ES negatives maximizes the ES and clinical data for both diagnoses and candidate gene identification, without GS in the majority. This iterative approach is cost-effective and is pertinent to the current conundrum of ES negatives.
Abstract
Sphingolipids are a diverse family of lipids with critical structural and signalling functions in the mammalian nervous system, where they are abundant in myelin membranes. Serine ...palmitoyltransferase, the enzyme that catalyses the rate-limiting reaction of sphingolipid synthesis, is composed of multiple subunits including an activating subunit, SPTSSA. Sphingolipids are both essential and cytotoxic and their synthesis must therefore be tightly regulated. Key to the homeostatic regulation are the ORMDL proteins that are bound to serine palmitoyltransferase and mediate feedback inhibition of enzymatic activity when sphingolipid levels become excessive.
Exome sequencing identified potential disease-causing variants in SPTSSA in three children presenting with a complex form of hereditary spastic paraplegia. The effect of these variants on the catalytic activity and homeostatic regulation of serine palmitoyltransferase was investigated in human embryonic kidney cells, patient fibroblasts and Drosophila.
Our results showed that two different pathogenic variants in SPTSSA caused a hereditary spastic paraplegia resulting in progressive motor disturbance with variable sensorineural hearing loss and language/cognitive dysfunction in three individuals. The variants in SPTSSA impaired the negative regulation of serine palmitoyltransferase by ORMDLs leading to excessive sphingolipid synthesis based on biochemical studies and in vivo studies in Drosophila.
These findings support the pathogenicity of the SPTSSA variants and point to excessive sphingolipid synthesis due to impaired homeostatic regulation of serine palmitoyltransferase as responsible for defects in early brain development and function.
Srivastava et al. identify monoallelic missense and biallelic frameshift variants in SPTSSA, encoding a subunit of serine palmitoyltransferase (SPT), in three individuals with a new complex hereditary spastic paraplegia. The variants disrupt ORMDL-mediated regulation of SPT resulting in excessive sphingolipid synthesis.
Here we describe a patient who presented with a history of congenital diaphragmatic hernia, inguinal hernia, and recurrent umbilical hernia. She also has joint laxity, hypotonia, and dysmorphic ...features. A unifying diagnosis was not identified based on her clinical phenotype. As part of her evaluation through the Undiagnosed Diseases Network, trio whole-exome sequencing was performed. Pathogenic variants in
and
were identified as causing two distinct autosomal dominant conditions, each with de novo inheritance. Fibrillin 1 (
mutations are associated with Marfan syndrome and a spectrum of similar phenotypes.
mutations are associated with trichorhinophalangeal syndrome types I and III. Features of both conditions are evident in the patient reported here. Discrepant features of the conditions (e.g., stature) and the young age of the patient may have made a clinical diagnosis more difficult in the absence of exome-wide genetic testing.
Abstract
DROSHA encodes a ribonuclease that is a subunit of the Microprocessor complex and is involved in the first step of microRNA (miRNA) biogenesis. To date, DROSHA has not yet been associated ...with a Mendelian disease. Here, we describe two individuals with profound intellectual disability, epilepsy, white matter atrophy, microcephaly and dysmorphic features, who carry damaging de novo heterozygous variants in DROSHA. DROSHA is constrained for missense variants and moderately intolerant to loss-of-function (o/e = 0.24). The loss of the fruit fly ortholog drosha causes developmental arrest and death in third instar larvae, a severe reduction in brain size and loss of imaginal discs in the larva. Loss of drosha in eye clones causes small and rough eyes in adult flies. One of the identified DROSHA variants (p.Asp1219Gly) behaves as a strong loss-of-function allele in flies, while another variant (p.Arg1342Trp) is less damaging in our assays. In worms, a knock-in that mimics the p.Asp1219Gly variant at a worm equivalent residue causes loss of miRNA expression and heterochronicity, a phenotype characteristic of the loss of miRNA. Together, our data show that the DROSHA variants found in the individuals presented here are damaging based on functional studies in model organisms and likely underlie the severe phenotype involving the nervous system.
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