Novel loss-of-function mutation in HERC2 is associated with severe developmental delay and paediatric lethality.

BACKGROUND: The HERC2 gene encodes a 527 kDa E3 ubiquitin protein ligase that has key roles in cell cycle regulation, spindle formation during mitosis, mitochondrial functions and DNA damage responses. It has essential roles during embryonic development, particularly for neuronal and muscular functions. To date, missense mutations in HERC2 have been associated with an autosomal recessive neurodevelopmental disorder with some phenotypical similarities to Angelman syndrome, and a homozygous deletion spanning HERC2 and OCA2 causing a more severe neurodevelopmental phenotype. METHODS AND RESULTS: We ascertained a consanguineous family with a presumed autosomal recessive severe neurodevelopmental disorder that leads to paediatric lethality. In affected individuals, we identified a homozygous HERC2 frameshift variant that results in a premature stop codon and complete loss of HERC2 protein. Functional characterisation of this variant in fibroblasts, from one living affected individual, revealed impaired mitochondrial network and function as well as disrupted levels of known interacting proteins such as XPA. CONCLUSION: This study extends the genotype-phenotype correlation for HERC2 variants to include a distinct lethal neurodevelopmental disorder, highlighting the importance of further characterisation for HERC2-related disorders.

Genetic and phenotypic characterization of complex hereditary spastic paraplegia.

The hereditary spastic paraplegias are a heterogeneous group of degenerative disorders that are clinically classified as either pure with predominant lower limb spasticity, or complex where spastic paraplegia is complicated with additional neurological features, and are inherited in autosomal dominant, autosomal recessive or X-linked patterns. Genetic defects have been identified in over 40 different genes, with more than 70 loci in total. Complex recessive spastic paraplegias have in the past been frequently associated with mutations in SPG11 (spatacsin), ZFYVE26/SPG15, SPG7 (paraplegin) and a handful of other rare genes, but many cases remain genetically undefined. The overlap with other neurodegenerative disorders has been implied in a small number of reports, but not in larger disease series. This deficiency has been largely due to the lack of suitable high throughput techniques to investigate the genetic basis of disease, but the recent availability of next generation sequencing can facilitate the identification of disease-causing mutations even in extremely heterogeneous disorders. We investigated a series of 97 index cases with complex spastic paraplegia referred to a tertiary referral neurology centre in London for diagnosis or management. The mean age of onset was 16 years (range 3 to 39). The SPG11 gene was first analysed, revealing homozygous or compound heterozygous mutations in 30/97 (30.9%) of probands, the largest SPG11 series reported to date, and by far the most common cause of complex spastic paraplegia in the UK, with severe and progressive clinical features and other neurological manifestations, linked with magnetic resonance imaging defects. Given the high frequency of SPG11 mutations, we studied the autophagic response to starvation in eight affected SPG11 cases and control fibroblast cell lines, but in our restricted study we did not observe correlations between disease status and autophagic or lysosomal markers. In the remaining cases, next generation sequencing was carried out revealing variants in a number of other known complex spastic paraplegia genes, including five in SPG7 (5/97), four in FA2H (also known as SPG35) (4/97) and two in ZFYVE26/SPG15 Variants were identified in genes usually associated with pure spastic paraplegia and also in the Parkinson's disease-associated gene ATP13A2, neuronal ceroid lipofuscinosis gene TPP1 and the hereditary motor and sensory neuropathy DNMT1 gene, highlighting the genetic heterogeneity of spastic paraplegia. No plausible genetic cause was identified in 51% of probands, likely indicating the existence of as yet unidentified genes.

Single-cell nanobiopsy enables multigenerational longitudinal transcriptomics of cancer cells.

Single-cell RNA sequencing has revolutionized our understanding of cellular heterogeneity, but routine methods require cell lysis and fail to probe the dynamic trajectories responsible for cellular state transitions, which can only be inferred. Here, we present a nanobiopsy platform that enables the injection of exogenous molecules and multigenerational longitudinal cytoplasmic sampling from a single cell and its progeny. The technique is based on scanning ion conductance microscopy (SICM) and, as a proof of concept, was applied to longitudinally profile the transcriptome of single glioblastoma (GBM) brain tumor cells in vitro over 72 hours. The GBM cells were biopsied before and after exposure to chemotherapy and radiotherapy, and our results suggest that treatment either induces or selects for more transcriptionally stable cells. We envision the nanobiopsy will contribute to transforming standard single-cell transcriptomics from a static analysis into a dynamic assay.

Proof-of-Concept Pilot Study on Comprehensive Spatiotemporal Intra-Patient Heterogeneity for Colorectal Cancer With Liver Metastasis.

Introduction: The mechanisms underlying high drug resistance and relapse rates after multi-modal treatment in patients with colorectal cancer (CRC) and liver metastasis (LM) remain poorly understood. Objective: We evaluate the potential translational implications of intra-patient heterogeneity (IPH) comprising primary and matched metastatic intratumor heterogeneity (ITH) coupled with circulating tumor DNA (ctDNA) variability. Methods: A total of 122 multi-regional tumor and perioperative liquid biopsies from 18 patients were analyzed via targeted next-generation sequencing (NGS). Results: The proportion of patients with ITH were 53% and 56% in primary CRC and LM respectively, while 35% of patients harbored de novo mutations in LM indicating spatiotemporal tumor evolution and the necessity of multiregional analysis. Among the 56% of patients with alterations in liquid biopsies, de novo mutations in cfDNA were identified in 25% of patients, which were undetectable in both CRC and LM. All 17 patients with driver alterations harbored mutations targetable by molecularly targeted drugs, either approved or currently under evaluation. Conclusion: Our proof-of-concept prospective study provides initial evidence on potential clinical superiority of IPH and warrants the conduction of precision oncology trials to evaluate the clinical utility of I PH-driven matched therapy.

Missense mutation of MAL causes a rare leukodystrophy similar to Pelizaeus-Merzbacher disease.

Leukodystrophies are a heterogenous group of genetic disorders, characterised by abnormal development of cerebral white matter. Pelizaeus-Merzbacher disease is caused by mutations in PLP1, encoding major myelin-resident protein required for myelin sheath assembly. We report a missense variant p.(Ala109Asp) in MAL as causative for a rare, hypomyelinating leukodystrophy similar to Pelizaeus-Merzbacher disease. MAL encodes a membrane proteolipid that directly interacts with PLP1, ensuring correct distribution during myelin assembly. In contrast to wild-type MAL, mutant MAL was retained in the endoplasmic reticulum but was released following treatment with 4-phenylbutyrate. Proximity-dependent identification of wild-type MAL interactants implicated post-Golgi vesicle-mediated protein transport and protein localisation to membranes, whereas mutant MAL interactants suggested unfolded protein responses. Our results suggest that mislocalisation of MAL affects PLP1 distribution, consistent with known pathomechanisms for hypomyelinating leukodystrophies.

Mitochondria as target to inhibit proliferation and induce apoptosis of cancer cells: the effects of doxycycline and gemcitabine.

Doxycycline has anti-tumour effects in a range of tumour systems. The aims of this study were to define the role mitochondria play in this process and examine the potential of doxycycline in combination with gemcitabine. We studied the adenocarcinoma cell line A549, its mitochondrial DNA-less derivative A549 ρ° and cultured fibroblasts. Treatment with doxycycline for 5 days resulted in a decrease of mitochondrial-encoded proteins, respiration and membrane potential, and an increase of reactive oxygen species in A549 cells and fibroblasts, but fibroblasts were less affected. Doxycycline slowed proliferation of A549 cells by 35%. Cellular ATP levels did not change. Doxycycline alone had no effect on apoptosis; however, in combination with gemcitabine given during the last 2 days of treatment, doxycycline increased caspase 9 and 3/7 activities, resulting in a further decrease of surviving A549 cells by 59% and of fibroblasts by 24% compared to gemcitabine treatment alone. A549 ρ° cells were not affected by doxycycline. Key effects of doxycycline observed in A549 cells, such as the decrease of mitochondrial-encoded proteins and surviving cells were also seen in the cancer cell lines COLO357 and HT29. Our results suggest that doxycycline suppresses cancer cell proliferation and primes cells for apoptosis by gemcitabine.