The rapid development in the last 10-15 years of microarray technologies, such as oligonucleotide
array Comparative Genomic Hybridization (CGH) and Single Nucleotide Polymorphisms
(SNP) genotyping ...array, has improved the identification of fine chromosomal structural variants, ranging
in length from kilobases (kb) to megabases (Mb), as an important cause of genetic differences
among healthy individuals and also as disease-susceptibility and/or disease-causing factors. Structural
genomic variations due to unbalanced chromosomal rearrangements are known as Copy-Number
Variants (CNVs) and these include variably sized deletions, duplications, triplications and translocations.
CNVs can significantly contribute to human diseases and rearrangements in several dosagesensitive
genes have been identified as an important causative mechanism in the molecular aetiology
of Charcot-Marie-Tooth (CMT) disease and of several CMT-related disorders, a group of inherited
neuropathies with a broad range of clinical phenotypes, inheritance patterns and causative genes. Duplications
or deletions of the dosage-sensitive gene PMP22 mapped to chromosome 17p12 represent
the most frequent causes of CMT type 1A and Hereditary Neuropathy with liability to Pressure Palsies
(HNPP), respectively. Additionally, CNVs have been identified in patients with other CMT types
(e.g., CMT1X, CMT1B, CMT4D) and different hereditary poly- (e.g., giant axonal neuropathy) and
focal- (e.g., hereditary neuralgic amyotrophy) neuropathies, supporting the notion of hereditary peripheral
nerve diseases as possible genomic disorders and making crucial the identification of fine
chromosomal rearrangements in the molecular assessment of such patients. Notably, the application of
advanced computational tools in the analysis of Next-Generation Sequencing (NGS) data has emerged
in recent years as a powerful technique for identifying a genome-wide scale complex structural variants
(e.g., as the ones resulted from balanced rearrangements) and also smaller pathogenic (intragenic)
CNVs that often remain beyond the detection limit of most conventional genomic microarray analyses;
in the context of inherited neuropathies where more than 70 disease-causing genes have been
identified to date, NGS and particularly Whole-Genome Sequencing (WGS) hold the potential to reduce
the number of genomic assays required per patient to reach a diagnosis, analyzing with a single
test all the Single Nucleotide Variants (SNVs) and CNVs in the genes possibly implicated in this heterogeneous
group of disorders.
Background:
Whether genetic factors influence the long-term course of multiple sclerosis (MS) is unresolved.
Objective:
To determine the influence of HLA-DRB1*1501 on long-term disease course in a ...homogeneous cohort of clinically isolated syndrome (CIS) patients.
Methods:
One hundred seven patients underwent clinical and MRI assessment at the time of CIS and after 1, 3, 5 and 15 years. HLA-DRB1*1501 status was determined using Sanger sequencing and tagging of the rs3135388 polymorphism. Linear/Poisson mixed-effects models were used to investigate rates of change in EDSS and MRI measures based on HLA-DRB1*1501 status.
Results:
HLA-DRB1*1501 -positive (n = 52) patients showed a faster rate of disability worsening compared with the HLA-DRB1*1501 -negative (n = 55) patients (annualised change in EDSS 0.14/year vs. 0.08/year, p < 0.025), and a greater annualised change in T2 lesion volume (adjusted difference 0.45 mL/year, p < 0.025), a higher number of gadolinium-enhancing lesions, and a faster rate of brain (adjusted difference −0.12%/year, p < 0.05) and spinal cord atrophy (adjusted difference −0.22 mm2/year, p < 0.05).
Interpretation:
These findings provide evidence that the HLA-DRB1*1501 allele plays a role in MS severity, as measured by long-term disability worsening and a greater extent of inflammatory disease activity and tissue loss. HLA-DRB1*1501 may provide useful information when considering prognosis and treatment decisions in early relapse-onset MS.
We report 2 families with undiagnosed recessive presynaptic congenital myasthenic syndrome (CMS). Whole exome or genome sequencing identified segregating homozygous variants in VAMP1: ...c.51_64delAGGTGGGGGTCCCC in a Kuwaiti family and c.146G>C in an Israeli family. VAMP1 is crucial for vesicle fusion at presynaptic neuromuscular junction (NMJ). Electrodiagnostic examination showed severely low compound muscle action potentials and presynaptic impairment. We assessed the effect of the nonsense mutation on mRNA levels and evaluated the NMJ transmission in VAMP1lew/lew mice, observing neurophysiological features of presynaptic impairment, similar to the patients. Taken together, our findings highlight VAMP1 homozygous mutations as a cause of presynaptic CMS. Ann Neurol 2017;81:597–603
Biallelic mutations in the gene that encodes the enzyme N-glycanase 1 (NGLY1) cause a rare disease with multi-symptomatic features including developmental delay, intellectual disability, neuropathy, ...and seizures. NGLY1’s activity in human neural cells is currently not well understood. To understand how NGLY1 gene loss leads to the specific phenotypes of NGLY1 deficiency, we employed direct conversion of NGLY1 patient-derived induced pluripotent stem cells (iPSCs) to functional cortical neurons. Transcriptomic, proteomic, and functional studies of iPSC-derived neurons lacking NGLY1 function revealed several major cellular processes that were altered, including protein aggregate-clearing functionality, mitochondrial homeostasis, and synaptic dysfunctions. These phenotypes were rescued by introduction of a functional NGLY1 gene and were observed in iPSC-derived mature neurons but not astrocytes. Finally, laser capture microscopy followed by mass spectrometry provided detailed characterization of the composition of protein aggregates specific to NGLY1-deficient neurons. Future studies will harness this knowledge for therapeutic development.
Display omitted
•NGLY1 neurons develop ProteoStat-positive aggregates that can be partially rescued•Profiling protein aggregates unveils links to other neurodegenerative diseases•NGLY1 phenotypes appear to be predominantly observed in neurons
Manole et al. employ cutting-edge techniques including laser capture microscopy to scrutinize the protein aggregates accumulating in NGLY1-deficient neurons. Their study unveils a significant enrichment of specific proteins associated with neurodegenerative diseases, providing novel insights into the pathological mechanisms underlying NGLY1 deficiency and shedding light on potential therapeutic targets.
Aptamer-functionalized biosensors exhibit high selectivity for monitoring neurotransmitters in complex environments. We translated nanoscale aptamer-modified nanopipette sensors to detect endogenous ...dopamine release in vitro and ex vivo. These sensors employ quartz nanopipettes with nanoscale pores (ca. 10 nm diameter) that are functionalized with aptamers that enable the selective capture of dopamine through target-specific conformational changes. The dynamic behavior of aptamer structures upon dopamine binding leads to the rearrangement of surface charge within the nanopore, resulting in measurable changes in ionic current. To assess sensor performance in real time, we designed a fluidic platform to characterize the temporal dynamics of nanopipette sensors. We then conducted differential biosensing by deploying control sensors modified with nonspecific DNA alongside dopamine-specific sensors in biological milieu. Our results confirm the functionality of aptamer-modified nanopipettes for direct measurements in undiluted complex fluids, specifically in the culture media of human-induced pluripotent stem cell-derived dopaminergic neurons. Moreover, sensor implantation and repeated measurements in acute brain slices was possible, likely owing to the protected sensing area inside nanoscale DNA-filled orifices, minimizing exposure to nonspecific interferents and preventing clogging. Further, differential recordings of endogenous dopamine released through electrical stimulation in the dorsolateral striatum demonstrate the potential of aptamer-modified nanopipettes for ex vivo recordings with unprecedented spatial resolution and reduced tissue damage.
The diverse clinical phenotypes of Wolf–Hirschhorn syndrome (WHS) are the result of haploinsufficiency of several genes, one of which, LETM1, encodes a protein of the mitochondrial inner membrane of ...uncertain function. Here, we show that LETM1 is associated with mitochondrial ribosomes, is required for mitochondrial DNA distribution and expression, and regulates the activity of an ancillary metabolic enzyme, pyruvate dehydrogenase. LETM1 deficiency in WHS alters mitochondrial morphology and DNA organization, as does substituting ketone bodies for glucose in control cells. While this change in nutrient availability leads to the death of fibroblasts with normal amounts of LETM1, WHS‐derived fibroblasts survive on ketone bodies, which can be attributed to their reduced dependence on glucose oxidation. Thus, remodeling of mitochondrial nucleoprotein complexes results from the inability of mitochondria to use specific substrates for energy production and is indicative of mitochondrial dysfunction. However, the dysfunction could be mitigated by a modified diet—for WHS, one high in lipids and low in carbohydrates.
Synopsis
The mitochondrial inner membrane protein LETM1 regulates mitochondrial DNA metabolism according to nutrient availability, which suggests that in the Wolf‐Hirschhorn syndrome and a range of other mitochondrial disorders, dietary control could impact disease development and progression.
Nutrients configure mitochondrial nucleoprotein complexes for energy production; for glucose such adaptation is mediated by LETM1.
LETM1 and pyruvate dehydrogenase (PDH) are physically associated with mitochondrial nucleoprotein complexes.
Monoallelic deletion of LETM1 inactivates PDH and causes aggregation of mitochondrial nucleoids.
LETM1 haploinsufficiency permits proliferation on a strict ketone body growth regime, whereas control cells undergo irreversible mitochondrial DNA aggregation and cell death.
Mitochondrial translation and ribosome maintenance in human cells requires LETM1 when the organelles utilize glucose and pyruvate.
The mitochondrial inner membrane protein LETM1 regulates mitochondrial DNA metabolism according to nutrient availability, which suggests that in the Wolf‐Hirschhorn syndrome and a range of other mitochondrial disorders, dietary control could impact disease development and progression.
Parkinson's disease (PD) is the second most prevalent neurodegenerative disease. Primary symptoms of PD arise with the loss of dopaminergic (DA) neurons in the Substantia Nigra Pars Compacta, but PD ...also affects the hippocampus and cortex, usually in its later stage. Approximately 15% of PD cases are familial with a genetic mutation. Two of the most associated genes with autosomal recessive (AR) early-onset familial PD are PINK1 and PRKN. In vitro studies of these genetic mutations are needed to understand the neurophysiological changes in patients' neurons that may contribute to neurodegeneration. In this work, we generated and differentiated DA and hippocampal neurons from human induced pluripotent stem cells (hiPSCs) derived from two patients with a double mutation in their PINK1 and PRKN (one homozygous and one heterozygous) genes and assessed their neurophysiology compared to two healthy controls. We showed that the synaptic activity of PD neurons generated from patients with the PINK1 and PRKN mutations is impaired in the hippocampus and dopaminergic neurons. Mutant dopaminergic neurons had enhanced excitatory post-synaptic activity. In addition, DA neurons with the homozygous mutation of PINK1 exhibited more pronounced electrophysiological differences compared to the control neurons. Signaling network analysis of RNA sequencing results revealed that Focal adhesion and ECM receptor pathway were the top two upregulated pathways in the mutant PD neurons. Our findings reveal that the phenotypes linked to PINK1 and PRKN mutations differ from those from other PD mutations, suggesting a unique interplay between these two mutations that drives different PD mechanisms.
Parkinson's disease (PD) is a neurodegenerative disease with both genetic and sporadic origins. In this study, we investigated the electrophysiological properties, synaptic activity, and gene ...expression differences in dopaminergic (DA) neurons derived from induced pluripotent stem cells (iPSCs) of healthy controls, sporadic PD (sPD) patients, and PD patients with E326K-GBA1 mutations. Our results demonstrate reduced sodium currents and synaptic activity in DA neurons derived from PD patients with E326K-GBA1 mutations, suggesting a potential contribution to PD pathophysiology. We also observed distinct electrophysiological alterations in sPD DA neurons, which included a decrease in synaptic currents. RNA sequencing analysis revealed unique dysregulated pathways in sPD neurons and E326K-GBA1 neurons, further supporting the notion that molecular mechanisms driving PD may differ between PD patients. In agreement with our previous reports, Extracellular matrix and Focal adhesion pathways were among the top dysregulated pathways in DA neurons from sPD patients and from patients with E326K-GBA1 mutations. Overall, our study further confirms that impaired synaptic activity is a convergent functional phenotype in DA neurons derived from PD patients across multiple genetic mutations as well as sPD. At the transcriptome level, we find that the brain extracellular matrix is highly involved in PD pathology across multiple PD-associated mutations as well as sPD.
Mutations in nuclear-encoded protein subunits of the mitochondrial ribosome are an increasingly recognised cause of oxidative phosphorylation system (OXPHOS) disorders. Among them, mutations in the
...MRPL44
gene, encoding a structural protein of the large subunit of the mitochondrial ribosome, have been identified in four patients with OXPHOS defects and early-onset hypertrophic cardiomyopathy with or without additional clinical features. A 23-year-old individual with cardiac and skeletal myopathy, neurological involvement, and combined deficiency of OXPHOS complexes in skeletal muscle was clinically and genetically investigated. Analysis of whole-exome sequencing data revealed a homozygous mutation in
MRPL44
(c.467 T > G), which was not present in the biological father, and a region of homozygosity involving most of chromosome 2, raising the possibility of uniparental disomy. Short-tandem repeat and genome-wide SNP microarray analyses of the family trio confirmed complete maternal uniparental isodisomy of chromosome 2. Mitochondrial ribosome assembly and mitochondrial translation were assessed in patient derived-fibroblasts. These studies confirmed that c.467 T > G affects the stability or assembly of the large subunit of the mitochondrial ribosome, leading to impaired mitochondrial protein synthesis and decreased levels of multiple OXPHOS components. This study provides evidence of complete maternal uniparental isodisomy of chromosome 2 in a patient with
MRPL44
-related disease, and confirms that
MRLP44
mutations cause a mitochondrial translation defect that may present as a multisystem disorder with neurological involvement.
Neuromuscular diseases are clinically and genetically heterogeneous and probably contain the greatest proportion of causative Mendelian defects than any other group of conditions. These disorders ...affect muscle and/or nerves with neonatal, childhood or adulthood onset, with significant disability and early mortality. Along with heterogeneity, unidentified and often very large genes require complementary and comprehensive methods in routine molecular diagnosis. Inevitably, this leads to increased diagnostic delays and challenges in the interpretation of genetic variants.
The application of next-generation sequencing, as a research and diagnostic strategy, has made significant progress into solving many of these problems. The analysis of these data is by no means simple, and the clinical input is essential to interpret results.
In this review, we describe using examples the recent advances in the genetic diagnosis of neuromuscular disorders, in research and clinical practice and the latest developments that are underway in next-generation sequencing. We also discuss the latest collaborative initiatives such as the Genomics England (Department of Health, UK) genome sequencing project that combine rare disease clinical phenotyping with genomics, with the aim of defining the vast majority of rare disease genes in patients as well as modifying risks and pharmacogenomics factors.
PDF
