A novel synonymous KMT2B variant in a patient with dystonia causes aberrant splicing

BACKGROUND: Heterozygous KMT2B variants are a common cause of dystonia. A novel synonymous KMT2B variant, c.5073C>T (p.Gly1691=) was identified in an individual with childhood-onset progressive dystonia. METHODS: The splicing impact of c.5073C>T was assessed using an in vitro exon-trapping assay. The genomic region of KMT2B exons 23-26 was cloned into the pSpliceExpress plasmid between exon 2 and 3 of the rat Ins2 gene. The c.5073C>T variant was then introduced through site-directed mutagenesis. The KMT2B wild-type and c.5073C>T plasmids were transfected separately into HeLa cells and RNA was extracted 48 hours after transfection. The RNA was reverse transcribed to produce cDNA, which was PCR amplified using primers annealing to the flanking rat Ins2 sequences. RESULTS: Sanger sequencing of the PCR products revealed that c.5073C>T caused a novel splice donor site and therefore a 5-bp deletion of KMT2B exon 23 in mature mRNA, leading to a coding frameshift and premature stop codon (p.Lys1692AsnfsTer7). CONCLUSION: To our knowledge, this is the first report of a KMT2B synonymous variant associated with dystonia. Reassessment of synonymous variants may increase diagnostic yield for inherited disorders including monogenic dystonia. This is of clinical importance, given the generally favourable response to deep brain stimulation for KMT2B-related dystonia

Comprehensive genetic diagnosis of tandem repeat expansion disorders with programmable targeted nanopore sequencing

More than 50 neurological and neuromuscular diseases are caused by short tandem repeat (STR) expansions, with 37 different genes implicated to date. We describe the use of programmable targeted long-read sequencing with Oxford Nanopore's ReadUntil function for parallel genotyping of all known neuropathogenic STRs in a single assay. Our approach enables accurate, haplotype-resolved assembly and DNA methylation profiling of STR sites, from a list of predetermined candidates. This correctly diagnoses all individuals in a small cohort (n = 37) including patients with various neurogenetic diseases (n = 25). Targeted long-read sequencing solves large and complex STR expansions that confound established molecular tests and short-read sequencing and identifies noncanonical STR motif conformations and internal sequence interruptions. We observe a diversity of STR alleles of known and unknown pathogenicity, suggesting that long-read sequencing will redefine the genetic landscape of repeat disorders. Last, we show how the inclusion of pharmacogenomic genes as secondary ReadUntil targets can further inform patient care

An inversion affecting the GCH1 gene as a novel finding in dopa-responsive dystonia

SUPPLEMENTARY FILE : SUPPLEMENTARY FIGURE S1. Pedigree of the family. Arrow indicates proband who underwent genetic studies, filled symbol indicates affected, squares represent males and circles represent females. d. MVA = died in motor vehicle accident. Additional family members were not available for testing. SUPPLEMENTARY FIGURE S2. Illumina short read whole genome sequencing data indicating a structural variant (NC_000014.8:g.[55343254_55346605del;55346606_60822142inv;60822143_60823119del]) on chromosome 14 affecting GCH1. Chimaeric (“split”) reads identified by the ClinSV tool are visualized in the IGV genome browser. To ease interpretation the alignments of segments of two representative reads are highlighted (A00488:195:HGJN7DSX2:3:2336:15573:12743 in red, and A00488:195:HGJN7DSX2:3:2160:31503:1219 in blue). Other tracks show the deletions which flank the inversion, and the exonic structure of GCH1 transcript NM_000161.3. (A) The left-hand (centromere proximal) breakpoint region, associated with a 3.4 kb deletion. (B) The right-hand (centromere distal) breakpoint region, associated with a smaller (1.0 kb) deletion. SUPPLEMENTARY FIGURE S3. Oxford Nanopore long read sequencing (LRS) data supporting the proposed structural variant. Chimaeric nanopore sequences are visualized in the IGV genome browser. The sequence alignments confirm the breakpoints indicated by short read sequencing analysis, and extend wide enough for a high level of confidence in read locations. (A, B) Inversion breakpoint regions as in Fig. S2, but in a 50 kb window.No abstract available.Paul Ainsworth Family Foundation. Open access publishing facilitated by University of New South Wales, as part of the Wiley - University of New South Wales agreement via the Council of Australian University Librarians.https://movementdisorders.onlinelibrary.wiley.com/journal/23301619hj2024School of Health Systems and Public Health (SHSPH)SDG-03:Good heatlh and well-bein

Monogenic Parkinson’s Disease: Genotype, Phenotype, Pathophysiology, and Genetic Testing

Parkinson’s disease may be caused by a single pathogenic variant (monogenic) in 5–10% of cases, but investigation of these disorders provides valuable pathophysiological insights. In this review, we discuss each genetic form with a focus on genotype, phenotype, pathophysiology, and the geographic and ethnic distribution. Well-established Parkinson’s disease genes include autosomal dominant forms (SNCA, LRRK2, and VPS35) and autosomal recessive forms (PRKN, PINK1 and DJ1). Furthermore, mutations in the GBA gene are a key risk factor for Parkinson’s disease, and there have been major developments for X-linked dystonia parkinsonism. Moreover, atypical or complex parkinsonism may be due to mutations in genes such as ATP13A2, DCTN1, DNAJC6, FBXO7, PLA2G6, and SYNJ1. Furthermore, numerous genes have recently been implicated in Parkinson’s disease, such as CHCHD2, LRP10, TMEM230, UQCRC1, and VPS13C. Additionally, we discuss the role of heterozygous mutations in autosomal recessive genes, the effect of having mutations in two Parkinson’s disease genes, the outcome of deep brain stimulation, and the role of genetic testing. We highlight that monogenic Parkinson’s disease is influenced by ethnicity and geographical differences, reinforcing the need for global efforts to pool large numbers of patients and identify novel candidate genes

Toll-Like Receptor-4 Inhibitor TAK-242 Attenuates Motor Dysfunction and Spinal Cord Pathology in an Amyotrophic Lateral Sclerosis Mouse Model

Neuroinflammation contributes to amyotrophic lateral sclerosis (ALS) progression. TLR4, a transmembrane protein that plays a central role in activation of the innate immune system, has been shown to induce microglial activation in ALS models. TLR4 is up-regulated in the spinal cords of hSOD1G93A mice. We aimed to examine the effects of specific TLR4 inhibition on disease progression and survival in the hSOD1G93A mouse model of ALS. Immunologic effect of TLR4 inhibition in vitro was measured by the effect of TAK-242 treatment on LPS-induced splenocytes proliferation. hSOD1G93A transgenic mice were treated with TAK-242, a selective TLR4 inhibitor, or vehicle. Survival, body weight, and motor behavior were monitored. To evaluate in vivo immunologic modifications associated with TAK-242 treatment, we measured serum IL-1β in the plasma, as well as IL-1β and TNF-α mRNAs in the spinal cord in wild-type mice and in TAK-242-treated and vehicle-treated early symptomatic hSOD1G93A mice. Immunohistochemical analysis of motor neurons, astrocytes, and microglial reactivity in the spinal cords were performed on symptomatic (100 days old) TAK-242-treated and vehicle-treated hSOD1G93A mice. In vitro, splenocytes taken from 100 days old hSOD1G93A mice showed significantly increased proliferation when exposed to LPS (p = 0.0002), a phenomenon that was reduced by TAK-242 (p = 0.0179). TAK-242 treatment did not attenuate body weight loss or significantly affect survival. However, TAK-242-treated hSOD1G93A mice showed temporary clinical delay in disease progression evident in the ladder test and hindlimb reflex measurements. Plasma IL-1β levels were significantly reduced in TAK-242-treated compared to vehicle-treated hSOD1G93A mice (p = 0.0023). TAK-242 treatment reduced spinal cord astrogliosis and microglial activation and significantly attenuated spinal cord motor neuron loss at early disease stage (p = 0.0259). Compared to wild-type animals, both IL-1β and TNF-α mRNAs were significantly upregulated in the spinal cords of hSOD1G93A mice. Spinal cord analysis in TAK-242-treated hSOD1G93A mice revealed significant attenuation of TNF-α mRNA (p = 0.0431), but no change in IL-1β mRNA. TLR4 inhibition delayed disease progression, attenuated spinal cord astroglial and microglial reaction, and reduced spinal motor neuron loss in the ALS hSOD1G93A mouse model. However, this effect did not result in increased survival. To our knowledge, this is the first report on TAK-242 treatment in a neurodegenerative disease model. Further studies are warranted to assess TLR4 as a therapeutic target in ALS