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Heterozygous variants in PRPF8 are associated with neurodevelopmental disorders
Abstract
The pre‐mRNA‐processing factor 8, encoded by PRPF8, is a scaffolding component of a spliceosome complex involved in the removal of introns from mRNA precursors. Previously, heterozygous pathogenic variants in PRPF8 have been associated with autosomal dominant retinitis pigmentosa. More recently, PRPF8 was suggested as a candidate gene for autism spectrum disorder due to the enrichment of sequence variants in this gene in individuals with neurodevelopmental disorders. We report 14 individuals with various forms of neurodevelopmental conditions, found to have heterozygous, predominantly de novo, missense, and loss‐of‐function variants in PRPF8. These individuals have clinical features that may represent a new neurodevelopmental syndrome.
About 15% of congenital heart disease (CHD) patients have a known pathogenic copy number variant. The majority of their chromosomal microarray (CMA) tests are deemed normal. Diagnostic interpretation typically ignores microdeletions smaller than 100 kb. We hypothesized that unreported microdeletions are enriched for CHD genes. We analyzed “normal” CMAs of 1762 patients who were evaluated at a pediatric referral center, of which 319 (18%) had CHD. Using CMAs from monozygotic twins or replicates from the same individual, we established a size threshold based on probe count for the reproducible detection of small microdeletions. Genes in the microdeletions were sequentially filtered by their nominal association with a CHD diagnosis, the expression level in the fetal heart, and the deleteriousness of a loss-of-function mutation. The subsequent enrichment for CHD genes was assessed using the presence of known or potentially novel genes implicated by a large whole-exome sequencing study of CHD. The unreported microdeletions were modestly enriched for both known CHD genes and those of unknown significance identified using their de novo mutation in CHD patients. Our results show that readily available “normal” CMA data can be a fruitful resource for genetic discovery and that smaller deletions should receive more attention in clinical evaluation.
Autism spectrum disorder (ASD) is among the most widespread neurodevelopmental diseases, with an approximate prevalence rate of 1 in 59. From a genetic point of view, this disorder is highly heterogeneous. This disorder is associated with both inheritable and de novo mutations in several genes. In addition to genetic loci that are identified through early karyotype analyses, recent advent of high throughput sequencing methods has facilitated identification of several genetic loci that confer risk of ASD. The current review provides an overview of different types of identified mutations including missense and nonsense mutations and copy number variations in various genes in individuals affected with ASD.
VEXAS (vacuoles, E1 enzyme, X-linked, autoinflammatory, somatic) syndrome is a new disease entity with autoinflammatory disorders (AID) driven by somatic variants in UBA1 that frequently co-exists with myelodysplastic syndromes (MDS). Clinicopathological and molecular features of Japanese cases with VEXAS-associated MDS remain elusive. We previously reported high prevalence of UBA1 variants in Japanese patients with relapsing polychondritis, in which 5 cases co-occurred with MDS. Here, we report clinicopathological and variant profiles of these 5 cases and 2 additional cases of MDS associated with VEXAS syndrome. Clinical characteristics of these cases included high prevalence of macrocytic anemia with marked cytoplasmic vacuoles in myeloid/erythroid precursors and low bone marrow (BM) blast percentages. All cases were classified as low or very low risk by the revised international prognostic scoring system (IPSS-R). Notably, 4 out of 7 cases showed significant improvement of anemia by treatment with prednisolone (PSL) or cyclosporin A (CsA), suggesting that an underlying inflammatory milieu induced by VEXAS syndrome may aggravate macrocytic anemia in VEXAS-associated MDS. Targeted deep sequencing of blood samples suggested that MDS associated with VEXAS syndrome tends to involve a smaller number of genes and lower risk genetic lesions than classical MDS.
Genetic variants that inactivate protein-coding genes are a powerful source of information about the phenotypic consequences of gene disruption: genes that are crucial for the function of an organism will be depleted of such variants in natural populations, whereas non-essential genes will tolerate their accumulation. However, predicted loss-of-function variants are enriched for annotation errors, and tend to be found at extremely low frequencies, so their analysis requires careful variant annotation and very large sample sizes1. Here we describe the aggregation of 125,748 exomes and 15,708 genomes from human sequencing studies into the Genome Aggregation Database (gnomAD). We identify 443,769 high-confidence predicted loss-of-function variants in this cohort after filtering for artefacts caused by sequencing and annotation errors. Using an improved model of human mutation rates, we classify human protein-coding genes along a spectrum that represents tolerance to inactivation, validate this classification using data from model organisms and engineered human cells, and show that it can be used to improve the power of gene discovery for both common and rare diseases. A catalogue of predicted loss-of-function variants in 125,748 whole-exome and 15,708 whole-genome sequencing datasets from the Genome Aggregation Database (gnomAD) reveals the spectrum of mutational constraints that affect these human protein-coding genes.
Purpose:
For neurodevelopmental disorders (NDDs), etiological evaluation can be a diagnostic odyssey involving numerous genetic tests, underscoring the need to develop a streamlined algorithm maximizing molecular diagnostic yield for this clinical indication. Our objective was to compare the yield of exome sequencing (ES) with that of chromosomal microarray (CMA), the current first-tier test for NDDs.
Methods:
We performed a PubMed scoping review and meta-analysis investigating the diagnostic yield of ES for NDDs as the basis of a consensus development conference. We defined NDD as global developmental delay, intellectual disability, and/or autism spectrum disorder. The consensus development conference included input from genetics professionals, pediatric neurologists, and developmental behavioral pediatricians.
Results:
After applying strict inclusion/exclusion criteria, we identified 30 articles with data on molecular diagnostic yield in individuals with isolated NDD, or NDD plus associated conditions (such as Rett-like features). Yield of ES was 36% overall, 31% for isolated NDD, and 53% for the NDD plus associated conditions. ES yield for NDDs is markedly greater than previous studies of CMA (15-20%).
Conclusion:
Our review demonstrates that ES consistently outperforms CMA for evaluation of unexplained NDDs. We propose a diagnostic algorithm placing ES at the beginning of the evaluation of unexplained NDDs.
At least six different proteins of the spliceosome, including PRPF3, PRPF4, PRPF6, PRPF8, PRPF31, and SNRNP200, are mutated in autosomal dominant retinitis pigmentosa (adRP). These proteins have recently been shown to localize to the base of the connecting cilium of the retinal photoreceptor cells, elucidating this form of RP as a retinal ciliopathy. In the case of loss-of-function variants in these genes, pathogenicity can easily be ascribed. In the case of missense variants, this is more challenging. Furthermore, the exact molecular mechanism of disease in this form of RP remains poorly understood. In this paper we take advantage of the recently published cryo EM-resolved structure of the entire human spliceosome, to predict the effect of a novel missense variant in one component of the spliceosome; PRPF31, found in a patient attending the genetics eye clinic at Bristol Eye Hospital. Monoallelic variants in PRPF31 are a common cause of autosomal dominant retinitis pigmentosa (adRP) with incomplete penetrance. We use in vitro studies to confirm pathogenicity of this novel variant PRPF31 c.341T > A, p.Ile114Asn. This work demonstrates how in silico modeling of structural effects of missense variants on cryo-EM resolved protein complexes can contribute to predicting pathogenicity of novel variants, in combination with in vitro and clinical studies. It is currently a considerable challenge to assign pathogenic status to missense variants in these proteins.
Background: Linking genotype to phenotype is a major aim of genetics research, yet the underlying biochemical mechanisms of many complex conditions continue to remain elusive. Recent research provides evidence that relevant gene-phenotype associations are discoverable in the study of intellectual disability (ID). Here we expand on that work, identifying distinctive gene interaction modules with unique enrichment patterns reflective of associated clinical features in ID.
Methods: Two hundred twelve forms of monogenic ID were curated according to comorbidities with autism and epilepsy. These groups were further subdivided according to secondary clinical manifestations of complex vs. simple facial dysmorphia and neurodegenerative-like features due to their clinical prominence, modest symptom overlap, and probable etiological divergence. An aggregate gene interaction ID network for these phenotype subgroups was discovered via a public database of known gene interactions: protein-protein, genetic, and mRNA coexpression. Additional annotation resources (Gene Ontology, Human Phenotype Ontology, TRANSFAC/JASPAR, and KEGG/WikiPathways) were utilized to assess functional and phenotypic enrichment patterns within subgroups.
Results: Phenotypic analysis revealed high rates of complex facial dysmorphia in ID with comorbid autism. In contrast, neurodegenerative-like features were overrepresented in ID with epilepsy. Network analysis subsequently showed that gene groups divided according to clinical features of interest resulted in distinctive interaction clusters, with unique functional enrichments according to gene set.
Conclusions: These data suggest that specific comorbid and secondary clinical features in ID are predictive of underlying genotype. In summary, ID form unique clusters, which are comprised of individual conditions with remarkable genotypic and phenotypic overlap.
Glaucoma is the cause of irreversible blindness worldwide. Mutations in six genes have been associated with juvenile- and adult-onset familial primary open angle glaucoma (POAG) prior to this report but they explain only a small proportion of the genetic load. The aim of the study is to identify the novel genetic cause of the POAG in the families with adult-onset glaucoma. Whole exome sequencing (WES) was performed on DNA of two affected individuals, and predicted pathogenic variants were evaluated for segregation in four affected and three unaffected Dutch family members by Sanger sequencing. We identified a pathogenic variant (p.Val956Gly) in the PRPF8 gene, which segregates with the disease in Dutch family. Targeted Sanger sequencing of PRPF8 in a panel of 40 POAG families (18 Pakistani and 22 Dutch) revealed two additional nonsynonymous variants (p.Pro13Leu and p.Met25Thr), which segregate with the disease in two other Pakistani families. Both variants were then analyzed in a case-control cohort consisting of Pakistani 320 POAG cases and 250 matched controls. The p.Pro13Leu and p.Met25Thr variants were identified in 14 and 20 cases, respectively, while they were not detected in controls (p values 0.0004 and 0.0001, respectively). Previously, PRPF8 mutations have been associated with autosomal dominant retinitis pigmentosa (RP). The PRPF8 variants associated with POAG are located at the N-terminus, while all RP-associated mutations cluster at the C-terminus, dictating a clear genotype-phenotype correlation.
Purpose:
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.
Methods:
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.
Results:
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.
Conclusion:
Our data support such a use of WES in pediatric neurology for disorders of presumed genetic origin.Genet Med advance online publication 23 March 2017Genetics in Medicine (2017); doi:10.1038/gim.2017.1.
A majority of human genes contain non-coding intervening sequences – introns that must be precisely excised from the pre-mRNA molecule. This event requires the coordinated action of five major small nuclear ribonucleoprotein particles (snRNPs) along with additional non-snRNP splicing proteins. Introns must be removed with nucleotidal precision, since even a single nucleotide mistake would result in a reading frame shift and production of a non-functional protein. Numerous human inherited diseases are caused by mutations that affect splicing, including mutations in proteins which are directly involved in splicing catalysis. One of the most common hereditary diseases associated with mutations in core splicing proteins is retinitis pigmentosa (RP). So far, mutations in more than 70 genes have been connected to RP. While the majority of mutated genes are expressed specifically in the retina, eight target genes encode for ubiquitous core snRNP proteins (Prpf3, Prpf4, Prpf6, Prpf8, Prpf31, and SNRNP200/Brr2) and splicing factors (RP9 and DHX38). Why mutations in spliceosomal proteins, which are essential in nearly every cell in the body, causes a disease that displays such a tissue-specific phenotype is currently a mystery. In this review, we recapitulate snRNP functions, summarize the missense mutations which are found in spliceosomal proteins as well as their impact on protein functions and discuss specific models which may explain why the retina is sensitive to these mutations.
Purpose:
We report the diagnostic yield of whole-exome sequencing (WES) in 3,040 consecutive cases at a single clinical laboratory.
Methods:
WES was performed for many different clinical indications and included the proband plus two or more family members in 76% of cases.
Results:
The overall diagnostic yield of WES was 28.8%. The diagnostic yield was 23.6% in proband-only cases and 31.0% when three family members were analyzed. The highest yield was for patients who had disorders involving hearing (55%, N = 11), vision (47%, N = 60), the skeletal muscle system (40%, N = 43), the skeletal system (39%, N = 54), multiple congenital anomalies (36%, N = 729), skin (32%, N = 31), the central nervous system (31%, N = 1,082), and the cardiovascular system (28%, N = 54). Of 2,091 cases in which secondary findings were analyzed for 56 American College of Medical Genetics and Genomics-recommended genes, 6.2% (N = 129) had reportable pathogenic variants. In addition to cases with a definitive diagnosis, in 24.2% of cases a candidate gene was reported that may later be reclassified as being associated with a definitive diagnosis.
Conclusion:
Our experience with our first 3,040 WES cases suggests that analysis of trios significantly improves the diagnostic yield compared with proband-only testing for genetically heterogeneous disorders and facilitates identification of novel candidate genes.Genet Med advance online publication 03 December 2015Genetics in Medicine (2015); doi:10.1038/gim.2015.148.
Disclaimer: These ACMG Standards and Guidelines were developed primarily as an educational resource for clinical laboratory geneticists to help them provide quality clinical laboratory services. Adherence to these standards and guidelines is voluntary and does not necessarily assure a successful medical outcome. These Standards and Guidelines should not be considered inclusive of all proper procedures and tests or exclusive of other procedures and tests that are reasonably directed to obtaining the same results. In determining the propriety of any specific procedure or test, the clinical laboratory geneticist should apply his or her own professional judgment to the specific circumstances presented by the individual patient or specimen. Clinical laboratory geneticists are encouraged to document in the patient’s record the rationale for the use of a particular procedure or test, whether or not it is in conformance with these Standards and Guidelines. They also are advised to take notice of the date any particular guideline was adopted and to consider other relevant medical and scientific information that becomes available after that date. It also would be prudent to consider whether intellectual property interests may restrict the performance of certain tests and other procedures.
The American College of Medical Genetics and Genomics (ACMG) previously developed guidance for the interpretation of sequence variants.¹ In the past decade, sequencing technology has evolved rapidly with the advent of high-throughput next-generation sequencing. By adopting and leveraging next-generation sequencing, clinical laboratories are now performing an ever-increasing catalogue of genetic testing spanning genotyping, single genes, gene panels, exomes, genomes, transcriptomes, and epigenetic assays for genetic disorders. By virtue of increased complexity, this shift in genetic testing has been accompanied by new challenges in sequence interpretation. In this context the ACMG convened a workgroup in 2013 comprising representatives from the ACMG, the Association for Molecular Pathology (AMP), and the College of American Pathologists to revisit and revise the standards and guidelines for the interpretation of sequence variants. The group consisted of clinical laboratory directors and clinicians. This report represents expert opinion of the workgroup with input from ACMG, AMP, and College of American Pathologists stakeholders. These recommendations primarily apply to the breadth of genetic tests used in clinical laboratories, including genotyping, single genes, panels, exomes, and genomes. This report recommends the use of specific standard terminology—“pathogenic,” “likely pathogenic,” “uncertain significance,” “likely benign,” and “benign”—to describe variants identified in genes that cause Mendelian disorders. Moreover, this recommendation describes a process for classifying variants into these five categories based on criteria using typical types of variant evidence (e.g., population data, computational data, functional data, segregation data). Because of the increased complexity of analysis and interpretation of clinical genetic testing described in this report, the ACMG strongly recommends that clinical molecular genetic testing should be performed in a Clinical Laboratory Improvement Amendments–approved laboratory, with results interpreted by a board-certified clinical molecular geneticist or molecular genetic pathologist or the equivalent.
Genet Med17 5, 405–423.
Mutations in genes that produce proteins involved in mRNA splicing, including pre-mRNA processing factors 3, 8, and 31 (PRPF3, 8, and 31), RP9, and SNRNP200 are common causes of the late-onset inherited blinding disorder retinitis pigmentosa (RP). It is not known how mutations in these ubiquitously expressed genes lead to retina-specific disease. To investigate the pathogenesis of the RNA splicing factor forms of RP, the authors generated and characterized the retinal phenotypes of Prpf3-T494M, Prpf8-H2309P knockin mice. The retinal ultrastructure of Prpf31-knockout mice was also investigated.
The knockin mice have single codon alterations in their endogenous Prpf3 and Prpf8 genes that mimic the most common disease causing mutations in human PRPF3 and PRPF8. The Prpf31-knockout mice mimic the null alleles that result from the majority of mutations identified in PRPF31 patients. The retinal phenotypes of the gene targeted mice were evaluated by electroretinography (ERG), light, and electron microscopy.
The RPE cells of heterozygous Prpf3(+/T494M) and Prpf8(+/H2309P) knockin mice exhibited loss of the basal infoldings and vacuolization, with accumulation of amorphous deposits between the RPE and Bruch[b]'s membrane at age two years. These changes were more severe in the homozygous mice, and were associated with decreased rod function in the Prpf3-T494M mice. Similar degenerative changes in the RPE were detected in Prpf31(±) mice at one year of age.
The finding of similar degenerative changes in RPE cells of all three mouse models suggests that the RPE may be the primary cell type affected in the RNA splicing factor forms of RP. The relatively late-onset phenotype observed in these mice is consistent with the typical adult onset of disease in patients with RP.
PRP8 protein of Saccharomyces cerevisiae interacts directly with pre-mRNA in spliceosomes, shown previously by UV-crosslinking. To analyse at which steps of splicing
and with which precursor-derived RNA species the interaction(s) take place, UV-crosslinking was combined with PRP8-specific
immunoprecipitation and the coprecipitated RNA species were analysed. Specific precipitation of intron-exon 2 and excised
intron species was observed. PRP8 protein could be UV-crosslinked to pre-mRNA in PRP2-depleted spliceosomes stalled before
initiation of the splicing reaction. Thus, the interaction of PRP8 protein with substrate RNA is established prior to the
first transesterification reaction, is maintained during both steps of splicing and continues with the excised intron after
completion of the splicing reaction. RNase T1 treatment of spliceosomes revealed that substrate RNA fragments of the 5′ splice
site region and the branchpoint-3′ splice site region could be coimmunoprecipitated with PRP8 specific antibodies, indicating
that these are potential sites of interaction for PRP8 protein with substrate RNA. Protection of the branch point-3′ splice
site region was detected only after step 1 of splicing. The results allow a first glimpse at the pattern of PRP8 protein-RNA
interactions during splicing and provide a fundamental basis for future analysis of these interactions.
This study reports the cloning, sequencing, and development of antisera against the human U5 snRNP 220-kDa protein or hPrp8p. Prp8p is the most highly conserved large nuclear protein known to date, but it is not related to any other protein. Southern, Northern, and expressed sequence tag analyses indicate that hPrp8p is encoded by a single gene. Prp8p is a core component of U5 snRNP and the U4/U6.U5 tri-snRNP, and antibodies raised against it immunoprecipitate both the major, U2-dependent and minor, U12-dependent spliceosomes. These spliceosomes, which excise different classes of introns, contain distinct sets of snRNAs overlapping only with U5 snRNA. Other than the core Sm proteins, hPrp8p is the first splicing factor shown to be common to both spliceosomes.
Retinitis pigmentosa (RP) is a genetically heterogeneous disorder characterized by progressive degeneration of the peripheral retina leading to night blindness and loss of visual fields. With an incidence of approximately 1 in 4000, RP can be inherited in X-linked, autosomal dominant or autosomal recessive modes. The RP13 locus for autosomal dominant RP (adRP) was placed on chromosome 17p13.3 by linkage mapping in a large South African adRP family. Using a positional cloning and candidate gene strategy, we have identified seven different missense mutations in the splicing factor gene PRPC8 in adRP families. Three of the mutations cosegregate within three RP13 linked families including the original large South African pedigree, and four additional mutations have been identified in other unrelated adRP families. The seven mutations are clustered within a 14 codon stretch within the last exon of this large 7 kb transcript. The altered amino acid residues at the C-terminus exhibit a high degree of conservation across species as diverse as humans, Arabidopsis and trypanosome, suggesting that some functional significance is associated with this part of the protein. These mutations in this ubiquitous and highly conserved splicing factor offer compelling evidence for a novel pathway to retinal degeneration.
Pre-messenger RNA (pre-mRNA) splicing is a central step in gene expression. Lying between transcription and protein synthesis, pre-mRNA splicing removes sequences (introns) that would otherwise disrupt the coding potential of intron-containing transcripts. This process takes place in the nucleus, catalyzed by a large RNA–protein complex called the spliceosome. Prp8p, one of the largest and most highly conserved of nuclear proteins, occupies a central position in the catalytic core of the spliceosome, and has been implicated in several crucial molecular rearrangements that occur there. Recently, Prp8p has also come under the spotlight for its role in the inherited human disease, Retinitis Pigmentosa.
Prp8 is unique, having no obvious homology to other proteins; however, using bioinformatical analysis we reveal the presence of a conserved RNA recognition motif (RRM), an MPN/JAB domain and a putative nuclear localization signal (NLS). Here, we review biochemical and genetical data, mostly related to the human and yeast proteins, that describe Prp8’s central role within the spliceosome and its molecular interactions during spliceosome formation, as splicing proceeds, and in post-splicing complexes.
Mutations in the systemically expressed pre-mRNA splicing-factor genes PRPF3, PRPF8, and PRPF31 have recently been associated with autosomal dominant retinitis pigmentosa (adRP). This study was intended to identify mutations in PRPF3, PRPF8, and PRPF31 in 150 Spanish families affected by adRP, to measure the contribution of mutations in these genes to adRP in that population, and to correlate RP phenotype expression with mutations in pre-mRNA splicing-factor genes.
Denaturing gradient gel electrophoresis (DGGE) and direct genomic sequencing were used to evaluate the complete coding region and flanking intronic sequences of the PRPF31 gene, exon 42 of PRPF8, and exon 11 of PRPF3 for mutations in 150 unrelated index patients with adRP. Ophthalmic and electrophysiological examination of patients with RP and their relatives was performed according to preexisting protocols.
Three nonsense mutations caused by insertion and deletion sequences and two missense mutations (Arg2310Gly) and within the stop codon of the PRPF8 gene (TGA-->TTG), were detected in five unrelated heterozygous patients. Three patients were heterozygous carriers of different nonsense mutations in exon 8 of the PRPF31, gene and one Thr494Met mutation was found in exon 11 of the PRPF3 gene. Cosegregation of the mutation in PRPF8 and PRPF3 with adRP was observed. However, two nonsense mutations in PRPF31 causing adRP detected in two families showed asymptomatic carriers.
Nine mutations, six of which are novel, in the pre-mRNA splicing-factor genes PRPF3, PRPF8, and PRPF31, causing adRP have been identified in the Spanish population. Their contribution to adRP is approximately 5% after correction in relation to mutations found in other genes causing adRP. The patients carrying a mutation in the pre-mRNA splicing-factor PRPF8 gene showed a type 1 diffuse RP. The existence of asymptomatic carriers of the nonsense mutation in the PRPF31 gene suggests incomplete penetrance for these mutations in the families.
Large genomic databases of neurodevelopmental disorders (NDD) are helpful resources of genomic variations in complex and heterogeneous conditions, as Autism Spectrum Disorder (ASD). We evaluated the role of rare copy number variations (CNVs) and exonic de novo variants, in a molecularly unexplored Brazilian cohort of 30 ASD trios (n = 90), by performing a meta‐analysis of our findings in more than 20,000 patients from NDD cohorts. We identified three pathogenic CNVs: two duplications on 1q21 and 17p13, and one deletion on 4q35. CNVs meta‐analysis (n = 8,688 cases and n = 3,591 controls) confirmed 1q21 relevance by identifying duplications in other 16 ASD patients. Exome analysis led the identification of seven de novo variants in ASD genes (SFARI list): three loss‐of‐function pathogenic variants in CUL3 , CACNA1H , and SHANK3 ; one missense pathogenic variant in KCNB1 ; and three deleterious missense variants in ATP10A , ANKS1B , and DOCK1 . From the remaining 12 de novo variants in non‐previous ASD genes, we prioritized PRPF8 and RBM14 . Meta‐analysis (n = 13,754 probands; n = 2,299 controls) identified six and two additional patients with validated de novo variants in PRPF8 and RBM14 , respectively. By comparing the de novo variants with a previously established mutational rate model, PRPF8 showed nominal significance before multiple test correction (P = 0.039, P ‐value adjusted = 0.079, binomial test), suggesting its relevance to ASD. Approximately 60% of our patients presented comorbidities, and the diagnostic yield was estimated in 23% (7/30: three pathogenic CNVs and four pathogenic de novo variants). Our uncharacterized Brazilian cohort with tetra‐hybrid ethnic composition was a valuable resource to validate and identify possible novel candidate loci. Autism Res 2020, 13: 199–206 . © 2019 International Society for Autism Research, Wiley Periodicals, Inc.
Lay Summary
We believed that to study an unexplored autistic population, such as the Brazilian, could help to find novel genes for autism. In order to test this idea, with our limited budget, we compared candidate genes obtained from genomic analyses of 30 children and their parents, with those of more than 20,000 individuals from international studies. Happily, we identified a genetic cause in 23% of our patients and suggest a possible novel candidate gene for autism (PRPF8 ).
ClinVar (https://www.ncbi.nlm.nih.gov/clinvar/) is a freely available, public archive of human genetic variants and interpretations of their significance to disease, maintained at the National Institutes of Health. Interpretations of the clinical significance of variants are submitted by clinical testing laboratories, research laboratories, expert panels and other groups. ClinVar aggregates data by variant-disease pairs, and by variant (or set of variants). Data aggregated by variant are accessible on the website, in an improved set of variant call format files and as a new comprehensive XML report. ClinVar recently started accepting submissions that are focused primarily on providing phenotypic information for individuals who have had genetic testing. Submissions may come from clinical providers providing their own interpretation of the variant ('provider interpretation') or from groups such as patient registries that primarily provide phenotypic information from patients ('phenotyping only'). ClinVar continues to make improvements to its search and retrieval functions. Several new fields are now indexed for more precise searching, and filters allow the user to narrow down a large set of search results.
Here we describe an overview and update on GeneMatcher (http://www.genematcher.org), a freely accessible web-based tool developed as part of the Baylor-Hopkins Center for Mendelian Genomics. We created GeneMatcher with the goal of identifying additional individuals with rare phenotypes who had variants in the same candidate disease gene. We also wanted to facilitate connections to basic scientists working on orthologous genes in model systems with the goal of connecting their work to human Mendelian phenotypes. Meeting these goals will enhance the identification of novel Mendelian genes. Launched in September 2013, GeneMatcher now has 2178 candidate genes from 486 submitters spread across 38 countries entered in the database (June 1(st) 2015). GeneMatcher is also part of the Matchmaker Exchange (http://matchmakerexchange.org/) with an Application Programing Interface enabling submitters to query other databases of genetic variants and phenotypes without having to create accounts and data entries in multiple systems. This article is protected by copyright. All rights reserved.
This article is protected by copyright. All rights reserved.
Mutated spliceosome components are recurrently being associated with perturbed tissue development and disease pathogenesis. Cephalophoˇnus (cph), is a zebrafish mutant carrying an early premature STOP codon in the spliceosome component Prpf8 (pre-mRNA processing factor 8). Cph initially develops normally, but then develops widespread cell death, especially in neurons, and is embryonic lethal. Cph mutants accumulate aberrantly spliced transcripts retaining both U2- and U12-type introns. Within early haematopoeisis, myeloid differentiation is impaired suggesting Prpf8 is required for haematopoietic development. Cph provides an animal model for zygotic PRPF8 dysfunction diseases and for evaluating therapeutic interventions.
The aim of this study was to report detailed genotype/phenotype correlation in two British autosomal dominant retinitis pigmentosa (adRP) families with recently described mutations in PRPF8.
Ten affected members from the two families (excluded for PRPF31 mutations) were assessed clinically. Seven subjects had fundus photography; some had electrophysiology, autofluorescence imaging, and visual field testing. Linkage analysis was performed from genomic DNA in one family. RNA was extracted from lymphocytes of the proband from both families, reverse transcribed into cDNA and subsequently screened for mutations in PRPF8. Segregation of mutations in each family was tested by direct genomic sequencing of the specific exons carrying the mutation.
All affected members complained of nyctalopia with variable age of onset. In the first family, there was marked variation in the clinical phenotype among affected individuals ranging from severe rod-cone dystrophy to a 67-year-old patient with a normal retinal appearance and mild rod dysfunction on scotopic electroretinography (ERG). The second family demonstrated similar variability and a history of a nonpenetrant individual. Linkage analysis in the first family showed strong evidence for linkage to markers on chromosome 17p implicating PRPF8 as a candidate gene. A c.6353 C>T change causing a nonconservative missense mutation p.S2118F was found in exon 38 of PRPF8 by direct sequencing of the cDNA. The mutation c.6930G>C (p.R2310S) was found in the second family.
This is the first report of marked intrafamilial variability associated with mutations in the PRPF8 gene, including incomplete penetrance. PRPF8 mutations should be suspected in patients with adRP and variable expressivity.
The yeast U4/U6.U5 pre-mRNA splicing small nuclear ribonucleoprotein (snRNP) is a 25S small nuclear ribonucleoprotein particle similar in size, composition, and morphology to its counterpart in human cells. The yeast U4/U6.U5 snRNP complex has been purified to near homogeneity by affinity chromatography and preparative glycerol gradient sedimentation. We show that there are at least 24 proteins stably associated with this particle and performed mass spectrometry microsequencing to determine their identities. In addition to the seven canonical core Sm proteins, there are a set of U6 snRNP specific Sm proteins, eight previously described U4/U6.U5 snRNP proteins, and four novel proteins. Two of the novel proteins have likely RNA binding properties, one has been implicated in the cell cycle, and one has no identifiable sequence homologues or functional motifs. The purification of the low abundance U4/U6.U5 snRNP from yeast and the powerful sequencing methodologies using small amounts of protein make possible the rapid identification of novel and previously unidentified components of large, low-abundance macromolecular machines from any genetically manipulable organism.
A Dutch family with autosomal dominant retinitis pigmentosa (adRP) displayed a phenotype characterized by an early age of onset, a diffuse loss of rod and cone sensitivity, and constricted visual fields (type I). One male showed a mild progression of the disease. Linkage analysis showed cosegregation of the genetic defect with markers from chromosome 17p13.1-p13.3, a region overlapping the RP13 locus. The critical interval of the RP locus as defined in this family was flanked by D17S926 and D17S786, with a maximal lod score of 4.2 (theta = 0.00) for marker D17S1529. Soon after the mapping of the underlying defect to the 17p13 region, a missense mutation (6970G>A; R2310K) was identified in exon 42 of the splicing factor gene PRPC8 in one patient of this family. Diagnostic restriction enzyme digestion of exon 42 amplified from genomic DNA of all family members revealed that the R2310K mutation segregated fully with the disease. The type I phenotype observed in this family is similar to that described for three other RP13 families with mutations in PRPC8.
Protein Prp8 interacts with several other spliceosomal proteins, snRNAs, and the pre-mRNA and thereby organizes the active site(s) of the spliceosome. The DEAD-box protein Brr2 and the GTPase Snu114 bind to the Prp8 C terminus, a region where mutations in human Prp8 are linked to the RP13 form of Retinitis pigmentosa. We show crystallographically that the C-terminal domain of yeast Prp8p exhibits a Jab1/MPN-like core known from deubiquitinating enzymes. Insertions and terminal appendices are grafted onto this core, covering a putative isopeptidase center whose metal binding site is additionally impaired. Targeted yeast-two-hybrid analyses show that the RP13-linked region in the C-terminal appendix of human Prp8 is essential for binding of human Brr2 and Snu114, and that RP13 point mutations in this fragment weaken these interactions. We conclude that the expanded Prp8 Jab1/MPN domain represents a pseudoenzyme converted into a protein-protein interaction platform and that dysfunction of this platform underlies Retinitis pigmentosa.
Nonsyndromic Retinitis Pigmentosa Overview
- A T Fahim
- S P Daiger