Novel deep intronic mutation in PLA2G6 causing early-onset Parkinson's disease with brain iron accumulation through pseudo-exon activation.
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ABSTRACT:
SUBMITTER: Cavestro C
PROVIDER: S-EPMC8426226 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Mutations of PLA2G6 gene are responsible for PARK14, an autosomal recessive L-DOPA responsive dystonia/parkinsonism with early/adult onset. This phenotype possesses an high clinical variability, which consists in the occurrence of cerebral and cerebellar atrophy, iron accumulation in the basal ganglia, and cognitive decline. This report describes a PD patient carrying an heterozygous PLA2G6 mutation, which was identified also in his PD affected sister. This patient is characterized by a L-DOPA responsive typical parkinsonian syndrome without the occurrence of dystonia, a slight cognitive decline, presence of iron accumulation both in neo and paleostriatum while cerebellar atrophy was absent. Clinical and imaging features are compatible with the PARK14 phenotype. Although PARK14 has been previously reported to be inherited as a recessive disorder, clinical and genetic analysis of this proband and his family rise the hypothesis that even heterozygous PLA2G6 mutations may cause PARK14. It remains to be analyzed whether these heterozygous variants may act as dominant mutations, or they merely increase the risk to develop PD by acting within a context of synergistic genetic and/or environmental backgrounds.
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Project description:Menkes disease is an X-linked disorder of copper metabolism caused by mutations in the ATP7A gene. Whereas most of the patients exhibit a severe classical form, about 9% of the patients exhibit a milder form of Menkes disease. The mildest form is called occipital horn syndrome (OHS). Mutations in the ATP7A gene can be identified in 95-98% of the Menkes disease patients by standard screening techniques. Investigation of RNA isolated from the fibroblasts of eleven patients with no identified mutations was performed, and revealed inclusion of new pseudo-exons into the ATP7A mRNA from three unrelated patients: two patients with OHS and one patient with classical Menkes disease. The pseudo-exons were inserted between exons 10 and 11, between exons 16 and 17 and between exons 14 and 15 in the three patients, as a result of deep intronic mutations. This is the first time the activation of pseudo-exons is demonstrated in the ATP7A gene, and it demonstrates the usefulness of RNA analysis, in terms of revealing disease-causing mutations in noncoding regions. The fact that three different mutations cause disease by the activation of pseudo-exon inclusion also indicates that in Menkes disease this is an important mechanism, which has hitherto been overlooked.
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Project description:BackgroundDeep-intronic variants that alter RNA splicing were ineffectively evaluated in the search for the cause of genetic diseases. Determination of such pathogenic variants from a vast number of deep-intronic variants (approximately 1,500,000 variants per individual) represents a technical challenge to researchers. Thus, we developed a Pathogenicity predictor for Deep-Intronic Variants causing Aberrant Splicing (PDIVAS) to easily detect pathogenic deep-intronic variants.ResultsPDIVAS was trained on an ensemble machine-learning algorithm to classify pathogenic and benign variants in a curated dataset. The dataset consists of manually curated pathogenic splice-altering variants (SAVs) and commonly observed benign variants within deep introns. Splicing features and a splicing constraint metric were used to maximize the predictive sensitivity and specificity, respectively. PDIVAS showed an average precision of 0.92 and a maximum MCC of 0.88 in classifying these variants, which were the best of the previous predictors. When PDIVAS was applied to genome sequencing analysis on a threshold with 95% sensitivity for reported pathogenic SAVs, an average of 27 pathogenic candidates were extracted per individual. Furthermore, the causative variants in simulated patient genomes were more efficiently prioritized than the previous predictors.ConclusionIncorporating PDIVAS into variant interpretation pipelines will enable efficient detection of disease-causing deep-intronic SAVs and contribute to improving the diagnostic yield. PDIVAS is publicly available at https://github.com/shiro-kur/PDIVAS .
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Project description:Wilson disease is a medically actionable rare autosomal recessive disorder of defective copper excretion caused by mutations in ATP7B, one of two highly evolutionarily conserved copper-transporting ATPases. Hundreds of disease-causing variants in ATP7B have been reported to public databases; more than half of these are missense changes, and a significant proportion are presumed unequivocal loss-of-function variants (nonsense, frameshift, and canonical splice site). Current molecular genetic testing includes sequencing all coding exons (±10 bp) as well as deletion/duplication testing, with reported sensitivity of >98%. We report a proband from a consanguineous family with a biochemical phenotype consistent with early-onset Wilson disease who tested negative on conventional molecular genetic testing. Using a combination of whole-genome sequencing and transcriptome sequencing, we found that the proband's disease is due to skipping of exons 6-7 of the ATP7B gene associated with a novel intronic variant (NM_000053.4:c.1947-19T > A) that alters a putative splicing enhancer element. This variant was also homozygous in the proband's younger sister, whose subsequent clinical evaluations revealed biochemical evidence of Wilson disease. Our work adds to emerging evidence that ATP7B exon skipping from deep intronic variants outside typical splice junctions is an important mechanism of Wilson disease; the variants responsible may elude standard genetic testing.
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Project description:Central core disease is a myopathy often arising from mutations in the type 1 ryanodine receptor (RYR1) gene, encoding the sarcoplasmic reticulum calcium release channel RyR1. No treatment is currently available for this disease. We studied the pathological situation of a severely affected child with two recessive mutations, which resulted in a massive reduction in the amount of RyR1. The paternal mutation induced the inclusion of a new in-frame pseudo-exon in RyR1 mRNA that resulted in the insertion of additional amino acids leading to the instability of the protein. We hypothesized that skipping this additional exon would be sufficient to restore RyR1 expression and to normalize calcium releases. We therefore developed U7-AON lentiviral vectors to force exon skipping on affected primary muscle cells. The efficiency of the exon skipping was evaluated at the mRNA level, at the protein level, and at the functional level using calcium imaging. In these affected cells, we observed a decreased inclusion of the pseudo-exon, an increased RyR1 protein expression, and a restoration of calcium releases of normal amplitude either upon direct RyR1 stimulation or in response to membrane depolarization. This study is the first demonstration of the potential of exon-skipping strategy for the therapy of central core disease, from the molecular to the functional level.
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Project description:PLA2G6 has been certified as a causative gene in patients with autosomal recessive early-onset Parkinson's disease (EOPD). We reported an EOPD case caused by PLA2G6 gene mutation, and performed neurological examination, genetic analysis, and multimodal neuroimaging to describe this phenotype. A compound heterozygous mutation c.991G>T/c.1472+1G>A was detected in this patient. Heterozygous for the c.991G>T and c.1472+1G>A were separately detected in his parents. Pathogenicity of these two mutations were predicted according to the American college of medical genetics and genomics (ACMG) guideline. MRI assessment showed absence of bilateral "swallow tail sign" and cerebellar atrophy in this patient, while no obvious difference in brain iron accumulation between PLA2G6 mutant PD patient and healthy controls. Cerebellar abnormalities may be a marker for diagnosis and evaluation of PLA2G6 mutation Parkinsonism. However, the iron accumulation in PD may not be the result of PLA2G6 mutation.
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Project description:Neurofibromatosis type 2 (NF2) is an autosomal-dominant disorder affecting about 1:33 000 newborns, mainly characterized by the development of tumors of the nervous system and ocular abnormalities. Around 85% of germline NF2 mutations are point mutations. Among them, ∼25% affect splicing and are associated with a variable disease severity. In the context of our NF2 Multidisciplinary Clinics, we have identified a patient fulfilling clinical criteria for the disease and exhibiting a severe phenotype. The patient carries a deep intronic mutation (g. 74409T>A, NG_009057.1) that produces the insertion of a cryptic exon of 167pb in the mature mRNA between exons 13 and 14, resulting in a truncated merlin protein (p.Pro482Profs*39). A mutation-specific antisense phosphorodiamidate morpholino oligomer was designed and used in vitro to effectively restore normal NF2 splicing in patient-derived primary fibroblasts. In addition, merlin protein levels were greatly recovered after morpholino treatment, decreasing patient's fibroblasts in vitro proliferation capacity and restoring cytoeskeleton organization. To our knowledge, this is the first NF2 case caused by a deep intronic mutation in which an in vitro antisense therapeutic approximation has been tested. These results open the possibility of using this approach in vivo for this type of mutation causing NF2.
| S-EPMC3722955 | biostudies-literature
Project description:The 2 major types of neurodegeneration with brain iron accumulation (NBIA) are the pantothenate kinase type 2 (PANK2)-associated neurodegeneration (PKAN) and NBIA2 or infantile neuroaxonal dystrophy (INAD) due to mutations in the phospholipase A2, group VI (PLA2G6) gene. We have recently demonstrated clinical heterogeneity in patients with mutations in the PLA2G6 gene by identifying a poorly defined subgroup of patients who present late with dystonia and parkinsonism. We report the clinical and genetic features of 7 cases with PLA2G6 mutations. Brain was available in 5 cases with an age of death ranging from 8 to 36 years and showed widespread alpha-synuclein-positive Lewy pathology, which was particularly severe in the neocortex, indicating that the Lewy pathology spread corresponded to Braak stage 6 and was that of the "diffuse neocortical type". In 3 cases there was hyperphosphorylated tau accumulation in both cellular processes as threads and neuronal perikarya as pretangles and neurofibrillary tangles. Later onset cases tended to have less tau involvement but still severe alpha-synuclein pathology. The clinical and neuropathological features clearly represent a link between PLA2G6 and parkinsonian disorders.
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Project description:Familial adenomatous polyposis (FAP) is a hereditary cancer syndrome that occurs as a result of germline mutations in the APC gene. Despite a clear clinical diagnosis of FAP, a certain proportion of the APC variants are not readily detectable through conventional genotyping routines. We accomplished genome sequencing in duo of the disease-affected proband and non-affected sibling followed by in silico predictions and a series of RNA-based assays clarifying variant functionality. By prioritizing variants obtained by genome sequencing, we discovered the novel deep intronic alteration APC:c.531 + 1482 A > G that was demonstrated to cause out-of-frame exonization of 56 base pairs from intron 5 of the gene. Further cDNA assays confirmed, that the aberrant splicing event was complete and its splice product was subject to nonsense-mediated decay. Co-segregation was observed between the variant carrier status and the disease phenotype. Cumulative evidence confirmed that APC:c.531 + 1482 A > G is a pathogenic variant causative of the disease.
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Project description:BackgroundOver half of children with rare genetic diseases remain undiagnosed despite maximal clinical evaluation and DNA-based genetic testing. As part of an Undiagnosed Diseases Program applying transcriptome (RNA) sequencing to identify the causes of these unsolved cases, we studied a child with severe infantile osteopetrosis leading to cranial nerve palsies, bone deformities, and bone marrow failure, for whom whole-genome sequencing was nondiagnostic.MethodsWe performed transcriptome (RNA) sequencing of whole blood followed by analysis of aberrant transcript isoforms and osteoclast functional studies.ResultsWe identified a pathogenic deep intronic variant in CLCN7 creating an unexpected, frameshifting pseudoexon causing complete loss of function. Functional studies, including osteoclastogenesis and bone resorption assays, confirmed normal osteoclast differentiation but loss of osteoclast function.ConclusionThis is the first report of a pathogenic deep intronic variant in CLCN7, and our approach provides a model for systematic identification of noncoding variants causing osteopetrosis-a disease for which molecular-genetic diagnosis can be pivotal for potentially curative hematopoietic stem cell transplantation. Our work illustrates that cryptic splice variants may elude DNA-only sequencing and supports broad first-line use of transcriptome sequencing for children with undiagnosed diseases.
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