Human Molecular Genetics
ISSN / EISSN : 0964-6906 / 1460-2083
Published by: Oxford University Press (OUP) (10.1093)
Total articles ≅ 13,120
Latest articles in this journal
Human Molecular Genetics; https://doi.org/10.1093/hmg/ddab295
Human cerebral cortical malformations are associated with progenitor proliferation and neuronal migration abnormalities. Progenitor cells include apical radial glia, intermediate progenitors and basal (or outer) radial glia (bRGs or oRGs). bRGs are few in number in lissencephalic species (e.g. the mouse) but abundant in gyrencephalic brains. The LIS1 gene coding for a dynein regulator, is mutated in human lissencephaly, associated also in some cases with microcephaly. LIS1 was shown to be important during cell division and neuronal migration. Here, we generated bRG-like cells in the mouse embryonic brain, investigating the role of Lis1 in their formation. This was achieved by in utero electroporation of a hominoid-specific gene TBC1D3 (coding for a RAB-GAP protein) at mouse embryonic day (E) 14.5. We first confirmed that TBC1D3 expression in wild-type (WT) brain generates numerous Pax6+ bRG-like cells that are basally localized. Second, using the same approach, we assessed the formation of these cells in heterozygote Lis1 mutant brains. Our novel results show that Lis1 depletion in the forebrain from E9.5 prevented subsequent TBC1D3-induced bRG-like cell amplification. Indeed, we observe perturbation of the ventricular zone (VZ) in the mutant. Lis1 depletion altered adhesion proteins and mitotic spindle orientations at the ventricular surface and increased the proportion of abventricular mitoses. Progenitor outcome could not be further altered by TBC1D3. We conclude that disruption of Lis1/LIS1 dosage is likely to be detrimental for appropriate progenitor number and position, contributing to lissencephaly pathogenesis.
Human Molecular Genetics; https://doi.org/10.1093/hmg/ddab299
Mutations in mitochondrial DNA encoded subunit of ATP synthase, MT-ATP6, are frequent causes of neurological mitochondrial diseases with a range of phenotypes from Leigh syndrome and NARP to ataxias and neuropathies. Here we investigated the functional consequences of an unusual heteroplasmic truncating mutation m.9154C>T in MT-ATP6, which caused peripheral neuropathy, ataxia and IgA nephropathy. ATP synthase not only generates cellular ATP, but its dimerization is required for mitochondrial cristae formation. Accordingly, the MT-ATP6 truncating mutation impaired the assembly of ATP synthase and disrupted cristae morphology, supporting our molecular dynamics simulations that predicted destabilized a/c subunit subcomplex. Next, we modeled the effects of the truncating mutation using patient-specific induced pluripotent stem cells. Unexpectedly, depending on mutation heteroplasmy level, the truncation showed multiple threshold effects in cellular reprogramming, neurogenesis and in metabolism of mature motor neurons (MN). Interestingly, MN differentiation beyond progenitor stage was impaired by Notch hyperactivation in the MT-ATP6 mutant, but not by rotenone-induced inhibition of mitochondrial respiration, suggesting that altered mitochondrial morphology contributed to Notch hyperactivation. Finally, we also identified a lower mutation threshold for a metabolic shift in mature MN, affecting lactate utilization, which may be relevant for understanding the mechanisms of mitochondrial involvement in peripheral motor neuropathies. These results establish a critical and disease-relevant role for ATP synthase in human cell fate decisions and neuronal metabolism.
Human Molecular Genetics; https://doi.org/10.1093/hmg/ddab296
Dominant GNAO1 mutations cause an emerging group of childhood-onset neurological disorders characterized by developmental delay, intellectual disability, movement disorders, drug-resistant seizures, and neurological deterioration. GNAO1 encodes the α-subunit of an inhibitory GTP/GDP-binding protein regulating ion channel activity and neurotransmitter release. The pathogenic mechanisms underlying GNAO1-related disorders remain largely elusive and there are no effective therapies. Here, we assessed the functional impact of two disease-causing variants associated with distinct clinical features, c.139A > G (p.S47G) and c.662C > A (p.A221D), using Caenorhabditis elegans as a model organism. The c.139A > G change was introduced into the orthologous position of the C. elegans gene via CRISPR/Cas9, whereas a knock-in strain carrying the p.A221D variant was already available. Like null mutants, homozygous knock-in animals showed increased egg laying and were hypersensitive to aldicarb, an inhibitor of acetylcholinesterase, suggesting excessive neurotransmitter release by different classes of motor neurons. Automated analysis of C. elegans locomotion indicated that goa-1 mutants move faster than control animals, with more frequent body bends and a higher reversal rate, and display uncoordinated locomotion. Phenotypic profiling of heterozygous animals revealed a strong hypomorphic effect of both variants, with a partial dominant-negative activity for the p.A221D allele. Finally, caffeine was shown to rescue aberrant motor function in C. elegans harboring the goa-1 variants; this effect is mainly exerted through adenosine receptor antagonism. Overall, our findings establish a suitable platform for drug discovery, which may assist in accelerating the development of new therapies for this devastating condition, and highlight the potential role of caffeine in controlling GNAO1-related dyskinesia.
Human Molecular Genetics; https://doi.org/10.1093/hmg/ddab297
Glycogen Storage Disease Type I (GSDI) is an inherited disease caused by glucose-6 phosphatase (G6Pase) deficiency, leading to a loss of endogenous glucose production and severe hypoglycemia. Moreover, most GSDI patients develop a chronic kidney disease (CKD) due to lipid accumulation in the kidney. Similar to diabetic CKD, activation of renin-angiotensin system (RAS) promotes renal fibrosis in GSDI. Here, we investigated the physiological and molecular effects of RAS blockers in GSDI patients and mice. A retrospective analysis of renal function was performed in 21 GSDI patients treated with RAS blockers. Cellular and metabolic impacts of RAS blockade were analyzed in K.G6pc−/− mice characterized by G6pc1 deletion in kidneys. GSDI patients started RAS blocker treatment at a median age of 21 years and long-term treatment reduced the progression of CKD in about 50% of patients. However, CKD progressed to kidney failure in 20% of treated patients, requiring renal transplantation. In K.G6pc−/− mice, CKD was associated with an impairment of autophagy and ER stress. RAS blockade resulted in a rescue of autophagy and decreased ER stress, concomitantly with decreased fibrosis and improved renal function, but without impact on glycogen and lipid contents. In conclusion, these data confirm the partial beneficial effect of RAS blockers in the prevention of CKD in GSDI. Mechanistically, we show that these effects are linked to a reduction of cell stress, without affecting metabolism.
Human Molecular Genetics; https://doi.org/10.1093/hmg/ddab294
Recent genome-wide association studies have identified 78 loci associated with Parkinson’s Disease susceptibility but the underlying mechanisms remain largely unclear. To identify variants likely causal for disease risk, we fine-mapped these Parkinson’s-associated loci using four different fine-mapping methods. We then integrated multi-assay cell-type-specific epigenomic profiles to pinpoint the likely mechanism of action of each variant, allowing us to identify Consensus SNPs that disrupt LRRK2 and FCGR2A regulatory elements in microglia, an MBNL2 enhancer in oligodendrocytes, and a DYRK1A enhancer in neurons. This genome-wide functional fine-mapping investigation of Parkinson’s Disease substantially advances our understanding of the causal mechanisms underlying this complex disease while avoiding focus on spurious, non-causal mechanisms. Together, these results provide a robust, comprehensive list of the likely causal variants, genes and cell-types underlying Parkinson’s Disease risk as demonstrated by consistently greater enrichment of our fine-mapped SNPs relative to lead GWAS SNPs across independent functional impact annotations. In addition, our approach prioritized an average of 3/85 variants per locus as putatively causal, making downstream experimental studies both more tractable and more likely to yield disease-relevant, actionable results.
Human Molecular Genetics; https://doi.org/10.1093/hmg/ddab293
The 10q24.33 locus is known to be associated with susceptibility to cutaneous malignant melanoma (CMM), but the mechanisms underlying this association have been not extensively investigated. We carried out an integrative genomic analysis of 10q24.33 using epigenomic annotations and in vitro reporter gene assays to identify regulatory variants. We found two putative functional single nucleotide polymorphisms (SNPs) in an enhancer and in the promoter of OBFC1, respectively, in neural crest and CMM cells, one, rs2995264, altering enhancer activity. The minor allele G of rs2995264 correlated with lower OBFC1 expression in 470 CMM tumors and was confirmed to increase the CMM risk in a cohort of 484 CMM cases and 1801 controls of Italian origin. Hi-C and chromosome conformation capture (3C) experiments showed the interaction between the enhancer-SNP region and the promoter of OBFC1 and an isogenic model characterized by CRISPR-Cas9 deletion of the enhancer-SNP region confirmed the potential regulatory effect of rs2995264 on OBFC1 transcription. Moreover, the presence of G-rs2995264 risk allele reduced the binding affinity of the transcription factor MEOX2. Biologic investigations showed significant cell viability upon depletion of OBFC1, specifically in CMM cells that were homozygous for the protective allele. Clinically, high levels of OBFC1 expression associated with histologically favorable CMM tumors. Finally, preliminary results suggested the potential effect of decreased OBFC1 expression on telomerase activity in tumorigenic conditions. Our results support the hypothesis that reduced expression of OBFC1 gene through functional heritable DNA variation can contribute to malignant transformation of normal melanocytes.
Human Molecular Genetics; https://doi.org/10.1093/hmg/ddab251
Glycogen synthase kinase 3 (GSK3) is a kinase mediating phosphorylation on serine and threonine amino acid residues of several target molecules. The enzyme is involved in the regulation of many cellular processes and aberrant activity of GSK3 has been linked to several disease conditions such as Fragile X Syndrome (FXS). Recent evidences demonstrating an increased activity of GSK3 in murine models of FXS, suggest that dysregulation/hyperactivation of the GSK3 path should contribute to FXS development. A likely possibility could be that in FXS there is a functional impairment of the upstream inhibitory input over GSK3 thus making overactive the kinase. Since GSK3 signaling is a central regulatory node for critical neurodevelopmental pathways, understanding the contribution of GSK3 dysregulation to FXS, may provide novel targets for therapeutic interventions for this disease. In this study we used AF3581, a potent GSK3 inhibitor that we recently discovered, in an in vivo FXS mouse model to elucidate the crucial role of GSK3 in specific behavioral patterns (locomotor activity, sensorimotor gating and social behavior) associated with this disease. All the behavioral alterations manifested by Fmr1 knockout mice were reverted after a chronic treatment with our GSK3 inhibitor, confirming the importance of this pathway as a therapeutic target.
Human Molecular Genetics; https://doi.org/10.1093/hmg/ddab279
Background Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a key player in lipid metabolism, as it degrades LDL receptors from hepatic cell membranes. So far, only variants of the PCSK9 gene locus were found to be associated with PCSK9 levels. Here we aimed to identify novel genetic loci that regulate PCSK9 levels and how they relate to other lipid traits. Additionally, we investigated to what extend the causal effect of PCSK9 on coronary artery disease (CAD) is mediated by LDL-C. Methods & Results We performed a genome-wide association study meta-analysis of PCSK9 levels in up to 12,721 samples of European ancestry. The estimated heritability was 10.3%, which increased to 12.6% using only samples from patients without statin treatment. We successfully replicated the known PCSK9 hit consisting of three independent signals. Interestingly, in a study of 300 African Americans, we confirmed the locus with a different PCSK9 variant. Beyond PCSK9, our meta-analysis detected three novel loci with genome-wide significance. Co-localization analysis with cis-eQTLs and lipid traits revealed biologically plausible candidate genes at two of them: APOB and TM6SF2. In a bivariate Mendelian Randomization analysis, we detected a strong effect of PCSK9 on LDL-C, but not vice versa. LDL-C mediated 63% of the total causal effect of PCSK9 on CAD. Conclusion Our study identified novel genetic loci with plausible candidate genes affecting PCSK9 levels. Ethnic heterogeneity was observed at the PCSK9 locus itself. While the causal effect of PCSK9 on CAD is mainly mediated by LDL-C, an independent direct effect also occurs.
Human Molecular Genetics; https://doi.org/10.1093/hmg/ddab283
Background Activated neutrophil-derived exosomes reportedly contribute to the proliferation of airway smooth muscle cells (ASMCs), thereby aggravating the airway wall remodeling during asthma; however, the specific mechanism remains unclear. Methods Lipopolysaccharide (LPS)-EXO and si-CRNDE-EXO were extracted from the media of human neutrophils treated with LPS and LPS + si-CRNDE (a siRNA targets long non-coding RNA CRNDE), respectively. Human ASMCs were co-cultured with LPS-EXO or si-CRNDE-EXO, and cell viability, proliferation, and migration were measured. The interplay of CRNDE, inhibitor of nuclear factor kappa B kinase subunit beta (IKKβ), and nuclear receptor subfamily 2 group C member 2 (TAK1) was explored using RNA immunoprecipitation (RIP) and Co-IP assays. A mouse model of asthma was induced using ovalbumin. Results CRNDE was upregulated in LPS-EXO and successfully transferred from LPS-treated neutrophils to ASMCs through exosome. Mechanically, CRNDE loaded in LPS-EXO reinforced TAK1-mediated IKKβ phosphorylation, thereby activating the nuclear factor kappa B (NF-κB) pathway. Functionally, silencing CRNDE in LPS-EXO, an IKKβ inhibitor, and an NF-κB inhibitor all removed the upregulation of cell viability, proliferation, and migration induced by LPS-EXO in ASMCs. In the end, the in vivo experiment demonstrated that CRNDE knockdown in neutrophils effectively reduced the thickness of bronchial smooth muscle in a mouse model for asthma. Conclusion Activated neutrophils-derived CRNDE was transferred to ASMCs through exosomes and activated the NF-κB pathway by enhancing IKKβ phosphorylation. The latter promoted the proliferation and migration of ASMCs and then contributed to airway remodeling in asthma.
Human Molecular Genetics; https://doi.org/10.1093/hmg/ddab289
The m.3243A>G mutation in the mitochondrial genome commonly causes retinal degeneration in patients with maternally inherited diabetes and deafness (MIDD) and mitochondrial encephalopathy, lactic acidosis, and stroke-like episodes (MELAS). Like other mitochondrial mutations, m.3243A>G is inherited from the mother with a variable proportion of wild type and mutant mitochondrial genomes in different cells. The mechanism by which the m.3243A>G variant in each tissue relates to the manifestation of disease phenotype is not fully understood. Using a digital PCR assay we found that the % m.3243G in skin derived dermal fibroblasts was positively correlated with that of blood from the same individual. The % m.3243G detected in fibroblast cultures remained constant over multiple passages and was negatively correlated with mtDNA copy number. Although the % m.3243G present in blood was not correlated with severity of vision loss, as quantified by Goldmann visual field, a significant negative correlation between % m.3243G and the age of onset of visual symptoms was detected. Together, these results indicate that precise measurement of % m.3243G in clinically accessible tissues such as skin and blood may yield information relevant to the management of retinal m.3243A>G associated disease.