Genomic technologies are transforming health care, with next-generation sequencing providing an important tool that underpins diagnostics, gene discovery, and the mechanistic understanding of ...disease.1 However, next-generation sequencing has had limited success in bringing the power of genomic medicine to neurological disorders caused by pathogenic repeat expansions.2 The development of research tools to identify repeat expansions in next-generation sequencing data has begun to address this deficit.3 In The Lancet Neurology, Kristina Ibañez and colleagues4 present compelling evidence of translation to clinical use, showing the utility of genome sequencing to diagnose neurogenetic repeat expansion disorders. ...diagnostic rates for repeat expansion disorders are low, with testing approaches being fragmented and incomplete. Similar outcomes are likely for the approximately 40 loci associated with repeat expansion neurological disorders not examined in this study, especially if pipelines implementing additional computational algorithms are used for consensus calling of expanded repeats.3 A current limitation of the software tools interrogating repeat expansion loci in short-read massively parallel sequence data is the inability to accurately determine sequence composition and size of larger repeat loci; however, these features can be examined using confirmatory testing with traditional methods or through future development of clinical-grade single molecule long-read next-generation sequencing.
The cytotoxicity of DNA-protein crosslinks (DPCs) is largely ascribed to their ability to block the progression of DNA replication. DPCs frequently occur in cells, either as a consequence of ...metabolism or exogenous agents, but the mechanism of DPC repair is not completely understood. Here, we characterize SPRTN as a specialized DNA-dependent and DNA replication-coupled metalloprotease for DPC repair. SPRTN cleaves various DNA binding substrates during S-phase progression and thus protects proliferative cells from DPC toxicity. Ruijs-Aalfs syndrome (RJALS) patient cells with monogenic and biallelic mutations in SPRTN are hypersensitive to DPC-inducing agents due to a defect in DNA replication fork progression and the inability to eliminate DPCs. We propose that SPRTN protease represents a specialized DNA replication-coupled DPC repair pathway essential for DNA replication progression and genome stability. Defective SPRTN-dependent clearance of DPCs is the molecular mechanism underlying RJALS, and DPCs are contributing to accelerated aging and cancer.
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•DNA-protein crosslinks (DPCs) stall DNA replication and induce genomic instability•SPARTAN (SPRTN) is a DNA replication-coupled metalloprotease which proteolyses DPCs•SPRTN metalloprotease is a fundamental enzyme in DPC repair pathway•Ruijs-Aalfs syndrome is caused by a defect in DPC repair due to mutations in SPRTN
Monogenic mutations in SPRTN cause genomic instability, premature aging, and hepatocellular carcinoma. The molecular mechanism of how SPRTN protects genome stability and prevents accelerated aging and cancer is not clear. Vaz, Popovic, et al. show that SPRTN is a DNA replication-coupled metalloprotease for DNA-protein crosslink repair in proliferative human cells.
Genomic technologies such as next-generation sequencing (NGS) are revolutionizing molecular diagnostics and clinical medicine. However, these approaches have proven inefficient at identifying ...pathogenic repeat expansions. Here, we apply a collection of bioinformatics tools that can be utilized to identify either known or novel expanded repeat sequences in NGS data. We performed genetic studies of a cohort of 35 individuals from 22 families with a clinical diagnosis of cerebellar ataxia with neuropathy and bilateral vestibular areflexia syndrome (CANVAS). Analysis of whole-genome sequence (WGS) data with five independent algorithms identified a recessively inherited intronic repeat expansion (AAGGG)exp in the gene encoding Replication Factor C1 (RFC1). This motif, not reported in the reference sequence, localized to an Alu element and replaced the reference (AAAAG)11 short tandem repeat. Genetic analyses confirmed the pathogenic expansion in 18 of 22 CANVAS-affected families and identified a core ancestral haplotype, estimated to have arisen in Europe more than twenty-five thousand years ago. WGS of the four RFC1-negative CANVAS-affected families identified plausible variants in three, with genomic re-diagnosis of SCA3, spastic ataxia of the Charlevoix-Saguenay type, and SCA45. This study identified the genetic basis of CANVAS and demonstrated that these improved bioinformatics tools increase the diagnostic utility of WGS to determine the genetic basis of a heterogeneous group of clinically overlapping neurogenetic disorders.
To describe a child meeting diagnostic criteria for tuberous sclerosis complex (TSC) carrying a pathogenic somatic variant in
, but no pathogenic variants in the 2 known TSC genes,
or
.
We present ...the clinical and imaging findings in a child presenting with drug-resistant focal seizures and multiple cortical tubers, a subependymal giant cell astrocytoma and multiple subependymal nodules in 1 cerebral hemisphere. Targeted panel sequencing and exome sequencing were performed on genomic DNA derived from blood and resected tuber tissue.
The child satisfied clinical diagnostic criteria for TSC, having 3 major features, only 2 of which are required for diagnosis. Genetic testing did not identify pathogenic variants or copy number variations in
or
but identified a pathogenic somatic
variant (NM_005614.4:c.104_105delACinsTA p.Tyr35Leu) in the cortical tuber.
RHEB is a partner of the TSC1/2 complex in the mechanistic target of rapamycin pathway. Somatic variants in
are associated with focal cortical dysplasia and hemimegalencephaly. We propose that variants in
may explain some of the genetically undiagnosed TSC cases and may be the third gene for TSC, or
.
Advances in understanding the etiology of Parkinson disease have been driven by the identification of causative mutations in families. Genetic analysis of an Australian family with three males ...displaying clinical features of early-onset parkinsonism and intellectual disability identified a ∼45 kb deletion resulting in the complete loss of RAB39B. We subsequently identified a missense mutation (c.503C>A p.Thr168Lys) in RAB39B in an unrelated Wisconsin kindred affected by a similar clinical phenotype. In silico and in vitro studies demonstrated that the mutation destabilized the protein, consistent with loss of function. In vitro small-hairpin-RNA-mediated knockdown of Rab39b resulted in a reduction in the density of α-synuclein immunoreactive puncta in dendritic processes of cultured neurons. In addition, in multiple cell models, we demonstrated that knockdown of Rab39b was associated with reduced steady-state levels of α-synuclein. Post mortem studies demonstrated that loss of RAB39B resulted in pathologically confirmed Parkinson disease. There was extensive dopaminergic neuron loss in the substantia nigra and widespread classic Lewy body pathology. Additional pathological features included cortical Lewy bodies, brain iron accumulation, tau immunoreactivity, and axonal spheroids. Overall, we have shown that loss-of-function mutations in RAB39B cause intellectual disability and pathologically confirmed early-onset Parkinson disease. The loss of RAB39B results in dysregulation of α-synuclein homeostasis and a spectrum of neuropathological features that implicate RAB39B in the pathogenesis of Parkinson disease and potentially other neurodegenerative disorders.
Dysembryoplastic neuroepithelial tumor (DNET) is a benign brain tumor associated with intractable drug-resistant epilepsy. In order to identify underlying genetic alterations and molecular ...mechanisms, we examined three family members affected by multinodular DNETs as well as 100 sporadic tumors from 96 patients, which had been referred to us as DNETs. We performed whole-exome sequencing on 46 tumors and targeted sequencing for hotspot
FGFR1
mutations and BRAF p.V600E was used on the remaining samples. FISH, copy number variation assays and Sanger sequencing were used to validate the findings. By whole-exome sequencing of the familial cases, we identified a novel germline
FGFR1
mutation, p.R661P. Somatic activating
FGFR1
mutations (p.N546K or p.K656E) were observed in the tumor samples and further evidence for functional relevance was obtained by in silico modeling. The FGFR1 p.K656E mutation was confirmed to be
in cis
with the germline p.R661P variant. In 43 sporadic cases, in which the diagnosis of DNET could be confirmed on central blinded neuropathology review, FGFR1 alterations were also frequent and mainly comprised intragenic tyrosine kinase
FGFR1
duplication and multiple mutants
in cis
(25/43; 58.1 %) while BRAF p.V600E alterations were absent (0/43). In contrast, in 53 cases, in which the diagnosis of DNET was not confirmed,
FGFR1
alterations were less common (10/53; 19 %;
p
< 0.0001) and hotspot BRAF p.V600E (12/53; 22.6 %) (
p
< 0.001) prevailed. We observed overexpression of phospho-ERK in FGFR1 p.R661P and p.N546K mutant expressing HEK293 cells as well as
FGFR1
mutated tumor samples, supporting enhanced MAP kinase pathway activation under these conditions. In conclusion, constitutional and somatic
FGFR1
alterations and MAP kinase pathway activation are key events in the pathogenesis of DNET. These findings point the way towards existing targeted therapies.
We describe first cousin sibling pairs with focal epilepsy, one of each pair having focal cortical dysplasia (FCD) IIa. Linkage analysis and whole‐exome sequencing identified a heterozygous germline ...frameshift mutation in the gene encoding nitrogen permease regulator‐like 3 (NPRL3). NPRL3 is a component of GAP Activity Towards Rags 1, a negative regulator of the mammalian target of rapamycin complex 1 signaling pathway. Immunostaining of resected brain tissue demonstrated mammalian target of rapamycin activation. Screening of 52 unrelated individuals with FCD identified 2 additional patients with FCDIIa and germline NPRL3 mutations. Similar to DEPDC5, NPRL3 mutations may be considered as causal variants in patients with FCD or magnetic resonance imaging–negative focal epilepsy. ANN NEUROL 2016;79:132–137
To determine the genetic basis of bottom-of-sulcus dysplasia (BOSD), which is a highly focal and epileptogenic cortical malformation in which the imaging, electrophysiologic, and pathologic ...abnormalities are maximal at the bottom of sulcus, tapering to a normal gyral crown.
Targeted panel deep sequencing (>500×) was performed on paired blood and brain-derived genomic DNA from 20 operated patients with drug-resistant focal epilepsy and BOSD. Histopathology was assessed using immunohistochemistry.
Brain-specific pathogenic somatic variants were found in 6 patients and heterozygous pathogenic germline variants were found in 2. Somatic variants were identified in
and germline variants were identified in
and
. Two patients with somatic
variants showed a mutation gradient, with higher mutation load at the bottom of sulcus compared to the gyral crown. Immunohistochemistry revealed an abundance of dysmorphic neurons and balloon cells in the bottom of sulcus but not in the gyral crown or adjacent gyri.
BOSD is associated with mTOR pathway dysregulation and shares common genetic etiologies and pathogenic mechanisms with other forms of focal and hemispheric cortical dysplasia, suggesting these disorders are on a genetic continuum.
Disturbed mitochondrial fusion and fission have been linked to various neurodegenerative disorders. In siblings from two unrelated families who died soon after birth with a profound ...neurodevelopmental disorder characterized by pontocerebellar hypoplasia and apnoea, we discovered a missense mutation and an exonic deletion in the SLC25A46 gene encoding a mitochondrial protein recently implicated in optic atrophy spectrum disorder. We performed functional studies that confirmed the mitochondrial localization and pro-fission properties of SLC25A46. Knockdown of slc24a46 expression in zebrafish embryos caused brain malformation, spinal motor neuron loss, and poor motility. At the cellular level, we observed abnormally elongated mitochondria, which was rescued by co-injection of the wild-type but not the mutant slc25a46 mRNA. Conversely, overexpression of the wild-type protein led to mitochondrial fragmentation and disruption of the mitochondrial network. In contrast to mutations causing non-lethal optic atrophy, missense mutations causing lethal congenital pontocerebellar hypoplasia markedly destabilize the protein. Indeed, the clinical severity appears inversely correlated with the relative stability of the mutant protein. This genotype-phenotype correlation underscores the importance of SLC25A46 and fine tuning of mitochondrial fission and fusion in pontocerebellar hypoplasia and central neurodevelopment in addition to optic and peripheral neuropathy across the life span.
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