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Zhong Nan Da Xue Xue Bao. Yi Xue Ban =... Oct 2023Hereditary neuropathy with liability to pressure palsy (HNPP) is a rare autosomal dominant peripheral neuropathy, usually caused by heterozygous deletion mutations in... (Review)
Review
OBJECTIVES
Hereditary neuropathy with liability to pressure palsy (HNPP) is a rare autosomal dominant peripheral neuropathy, usually caused by heterozygous deletion mutations in the peripheral myelin protein 22 () gene. This study aims to investigate the clinical and molecular genetic characteristics of HNPP.
METHODS
HNPP patients in the Department of Neurology at Third Xiangya Hospital of Central South University from 2009 to 2023 were included in this study. The general clinical data, nervous electrophysiological and molecular genetic examination results were collected and analyzed. Molecular genetic examination was to screen for deletion of gene using multiplex ligation-dependent probe amplification (MLPA) after extracting genomic DNA from peripheral blood; and if no deletion mutation was detected, next-generation sequencing was used to screen for point mutations. The related literatures of HNPP were reviewed, and the clinical and molecular genetic characteristics of HNPP patients were analyzed.
RESULTS
A total of 34 HNPP patients from 24 unrelated Chinese Han families were included in this study, including 25 males and 9 females. The average age at illness onset was 22.0 years. Sixty-two point five percent of the families had a positive family history. Among them, 30 patients had symptoms of peripheral nerve paralysis. Patients often presented with paroxysmal single limb weakness with (or) numbness (25/30), and some patients had paroxysmal unilateral recurrent laryngeal nerve (vagus nerve) paralysis (2/30). Physical examination revealed muscle weakness (23/29), hypoesthesia (9/29), weakened or absent ankle reflexes (20/29), distal limb muscle atrophy (8/29) and high arched feet (5/29). Most patients (26/30) could fully recover to normal after an acute attack. Thirty-one patients in our group underwent nervous electrophysiological examination, and showed multiple demyelinating peripheral neuropathies with both motor and sensory nerves involved. Most patients showed significantly prolonged distal motor latency (DML), mild to moderate nerve conduction velocity slowing, decreased amplitude of compound muscle action potential (CMAP) and sensory nerve action potential (SNAP), and sometimes with conduction block. Nerve motor conduction velocity was (48.5±5.5) m/s, and the CMAP amplitude was (8.4±5.1) mV. Nerve sensory conduction velocity was (37.4±10.5) m/s, and the SNAP amplitude was (14.4±15.2) μV. There were 24 families, 23 of whom had the classical deletion, the last one had a heterozygous pathogenic variant in the gene sequence (c.434delT). By reviewing clinical data and genetic testing results of reported 1 734 HNPP families, we found that heterozygous deletion mutation of was the most common pathogenic mutation of HNPP (93.4%). Other patients were caused by small mutations (4.0%), heterozygous gross deletions (0.6%), and complex rearrangements (0.1%). Thirty-eight sorts of HNPP-related small mutations was reported, including missense mutations (10/38), nonsense mutations (4/38), base deletion mutations (13/38), base insertion mutations (3/38), and shear site mutations (8/38). HNPP patients most often presented with episodic painless single nerve palsy. Common peroneal nerve, ulnar nerve, and brachial plexus nerve were the most common involved nerves, accounting for about 75%. Only eighteen patients with cranial nerve involved was reported.
CONCLUSIONS
Heterozygous deletion mutation of is the most common pathogenic mutation of HNPP. Patients is characterized by episodic and painless peripheral nerve paralysis, mainly involving common peroneal nerve, ulnar nerve, and other peripheral nerves. Nervous electrophysiological examination has high sensitivity and specificity for the diagnosis of HNPP, which is manifested by extensive demyelinating changes. For patients with suspected HNPP, nervous electrophysiological examination and -MLPA detection are preferred. Sanger sequencing or next generation sequencing can be considered to detect other mutations of .
Topics: Female; Male; Humans; Young Adult; Adult; Peripheral Nervous System Diseases; Paralysis; Genetic Testing; Molecular Biology; Arthrogryposis; Hereditary Sensory and Motor Neuropathy
PubMed: 38432886
DOI: 10.11817/j.issn.1672-7347.2023.230116 -
The Journal of Biological Chemistry Apr 2024Both POLG and MGME1 are needed for mitochondrial DNA (mtDNA) maintenance in animal cells. POLG, the primary replicative polymerase of the mitochondria, has an...
Both POLG and MGME1 are needed for mitochondrial DNA (mtDNA) maintenance in animal cells. POLG, the primary replicative polymerase of the mitochondria, has an exonuclease activity (3'→5') that corrects for the misincorporation of bases. MGME1 serves as an exonuclease (5'→3'), producing ligatable DNA ends. Although both have a critical role in mtDNA replication and elimination of linear fragments, these mechanisms are still not fully understood. Using digital PCR to evaluate and compare mtDNA integrity, we show that Mgme1 knock out (Mgme1 KK) tissue mtDNA is more fragmented than POLG exonuclease-deficient "Mutator" (Polg MM) or WT tissue. In addition, next generation sequencing of mutant hearts showed abundant duplications in/nearby the D-loop region and unique 100 bp duplications evenly spaced throughout the genome only in Mgme1 KK hearts. However, despite these unique mtDNA features at steady-state, we observed a similar delay in the degradation of mtDNA after an induced double strand DNA break in both Mgme1 KK and Polg MM models. Lastly, we characterized double mutant (Polg MM/Mgme1 KK) cells and show that mtDNA cannot be maintained without at least one of these enzymatic activities. We propose a model for the generation of these genomic abnormalities which suggests a role for MGME1 outside of nascent mtDNA end ligation. Our results highlight the role of MGME1 in and outside of the D-loop region during replication, support the involvement of MGME1 in dsDNA degradation, and demonstrate that POLG EXO and MGME1 can partially compensate for each other in maintaining mtDNA.
Topics: Animals; Mice; DNA Polymerase gamma; DNA Replication; DNA, Mitochondrial; DNA-Directed DNA Polymerase; Mice, Knockout
PubMed: 38432635
DOI: 10.1016/j.jbc.2024.107128 -
STAR Protocols Mar 2024RNA-DNA covalent hybrids (RDHs) are widely employed in biology. Although RDHs can be manufactured, the synthesis of molecules longer than 120 nucleotides is challenging....
RNA-DNA covalent hybrids (RDHs) are widely employed in biology. Although RDHs can be manufactured, the synthesis of molecules longer than 120 nucleotides is challenging. Here, we present a protocol for the generation and purification of high-grade purified high-molecular-weight 5'-RNA-DNA-3' hybrids. We describe steps for preparing oligos and buffers, ligation reaction, and high-performance liquid chromatography-based RDH purification. This protocol is executable in standard molecular biology laboratories.
Topics: RNA; DNA; RNA Ligase (ATP)
PubMed: 38430520
DOI: 10.1016/j.xpro.2024.102930 -
DNA Repair Apr 2024DNA polymerases lambda (Polλ) and mu (Polμ) are X-Family polymerases that participate in DNA double-strand break (DSB) repair by the nonhomologous end-joining pathway...
DNA polymerases lambda (Polλ) and mu (Polμ) are X-Family polymerases that participate in DNA double-strand break (DSB) repair by the nonhomologous end-joining pathway (NHEJ). Both polymerases direct synthesis from one DSB end, using template derived from a second DSB end. In this way, they promote the NHEJ ligation step and minimize the sequence loss normally associated with this pathway. The two polymerases differ in cognate substrate, as Polλ is preferred when synthesis must be primed from a base-paired DSB end, while Polμ is required when synthesis must be primed from an unpaired DSB end. We generated a Polλ variant (Polλ) that retained canonical Polλ activity on a paired end-albeit with reduced incorporation fidelity. We recently discovered that the variant had unexpectedly acquired the activity previously unique to Polμ-synthesis from an unpaired primer terminus. Though the sidechains of the Loop1 region make no contact with the DNA substrate, Polλ Loop1 amino acid sequence is surprisingly essential for its unique activity during NHEJ. Taken together, these results underscore that the Loop1 region plays distinct roles in different Family X polymerases.
Topics: DNA-Directed DNA Polymerase; Gain of Function Mutation; DNA Polymerase beta; DNA Repair; DNA; DNA End-Joining Repair
PubMed: 38428373
DOI: 10.1016/j.dnarep.2024.103645 -
Archives of Endocrinology and Metabolism Feb 2024Beckwith-Wiedemann syndrome (BWS) is a common genetic congenital disease characterized by somatic overgrowth and its broad clinical spectrum includes pre- and post-natal...
Beckwith-Wiedemann syndrome (BWS) is a common genetic congenital disease characterized by somatic overgrowth and its broad clinical spectrum includes pre- and post-natal macrosomia, macroglossia, visceromegaly, increased risk of neonatal hypoglycemia, and development of embryonic tumors. BWS occurs due to genetic/epigenetic changes involving growth-regulating genes, located on region 11p15, with an important genotype-phenotype correlation. Congenital adrenal hyperplasia (CAH) comprises a spectrum of autosomal recessive diseases presenting a variety of clinical manifestations due to a deficiency in one of the enzymes involved in cortisol secretion. Early diagnosis based on newborn screening prevents the adrenal crisis and early infant death. However, high 17-hydroxyprogesterone (17-OHP) levels can occur in newborns or premature infants without CAH, in situations of stress due to maternal or neonatal factors. Here, we report new cases of false-positive diagnosis of 21-hydroxylase deficiency during newborn screening - two girls and one boy with BWS. Methylation-specific multiplex ligation-dependent probe amplification revealed a gain of methylation in the H19 differentially methylated region. Notably, all three cases showed a complete normalization of biochemical changes, highlighting the transient nature of these hormonal findings that imitate the classical form of CAH. This report sheds light on a new cause of false-positive 21-hydroxylase deficiency diagnosis during newborn screening: Beckwith-Wiedemann syndrome.
Topics: Male; Infant; Female; Humans; Infant, Newborn; Beckwith-Wiedemann Syndrome; Adrenal Hyperplasia, Congenital; DNA Methylation; Neonatal Screening
PubMed: 38427811
DOI: 10.20945/2359-4292-2022-0395 -
Nature Mar 2024Cyclic GMP-AMP synthase (cGAS) senses aberrant DNA during infection, cancer and inflammatory disease, and initiates potent innate immune responses through the synthesis...
Cyclic GMP-AMP synthase (cGAS) senses aberrant DNA during infection, cancer and inflammatory disease, and initiates potent innate immune responses through the synthesis of 2'3'-cyclic GMP-AMP (cGAMP). The indiscriminate activity of cGAS towards DNA demands tight regulatory mechanisms that are necessary to maintain cell and tissue homeostasis under normal conditions. Inside the cell nucleus, anchoring to nucleosomes and competition with chromatin architectural proteins jointly prohibit cGAS activation by genomic DNA. However, the fate of nuclear cGAS and its role in cell physiology remains unclear. Here we show that the ubiquitin proteasomal system (UPS) degrades nuclear cGAS in cycling cells. We identify SPSB3 as the cGAS-targeting substrate receptor that associates with the cullin-RING ubiquitin ligase 5 (CRL5) complex to ligate ubiquitin onto nuclear cGAS. A cryo-electron microscopy structure of nucleosome-bound cGAS in a complex with SPSB3 reveals a highly conserved Asn-Asn (NN) minimal degron motif at the C terminus of cGAS that directs SPSB3 recruitment, ubiquitylation and cGAS protein stability. Interference with SPSB3-regulated nuclear cGAS degradation primes cells for type I interferon signalling, conferring heightened protection against infection by DNA viruses. Our research defines protein degradation as a determinant of cGAS regulation in the nucleus and provides structural insights into an element of cGAS that is amenable to therapeutic exploitation.
Topics: Animals; Humans; Mice; Cell Nucleus; Cryoelectron Microscopy; Degrons; DNA Virus Infections; DNA Viruses; DNA, Viral; Immunity, Innate; Innate Immunity Recognition; Interferon Type I; Nuclear Proteins; Nucleosomes; Nucleotidyltransferases; Proteasome Endopeptidase Complex; Protein Stability; Proteolysis; Substrate Specificity; Ubiquitin; Ubiquitin-Protein Ligases; Ubiquitination
PubMed: 38418882
DOI: 10.1038/s41586-024-07112-w -
Acta Myologica : Myopathies and... 2023Fukutin-related protein (FKRP) mutations cause a broad spectrum of muscular dystrophies, from a relatively mild limb-girdle muscular dystrophy type 9 (LGMDR9) to severe...
Fukutin-related protein (FKRP) mutations cause a broad spectrum of muscular dystrophies, from a relatively mild limb-girdle muscular dystrophy type 9 (LGMDR9) to severe congenital muscular dystrophy (CMD). This study aims to report two siblings belonging to a non-consanguineous Tunisian family harboring a novel compound heterozygous variant and presenting a mild LGDMR9 phenotype. For mutation screening, massive parallel sequencing was performed, followed by Sanger sequencing and multiplex ligation-dependent probe amplification (MLPA) to validate the existence of the discovered variants. The absence of alpha-dystroglycan was determined by immunohistochemistry. Brain and thigh magnetic resonance imaging (MRI) were performed to detect thigh and brain abnormalities. The two siblings had a late age at onset and clinical examination showed that the pelvic girdles had a predominantly proximal and symmetrical distribution of weakness without cardiac or respiratory involvement. They both had a modified Gardner-Medwin Walton Scale mGMWS grade of 4 and a modified Rankin Scale (mRS) score of 1. The DNA sequencing revealed a novel deletion of exons 2 and 3 in one allele and a missense mutation c.1364C > A, which has been reported to be responsible for congenital muscular dystrophy and mental retardation on the second allele. The simultaneous presence of the two variations in the two cases suggests that the variants segregate with the pathophysiology.
Topics: Humans; Muscle, Skeletal; Muscular Dystrophies; Muscular Dystrophies, Limb-Girdle; Mutation; Pentosyltransferases; Phenotype; Proteins
PubMed: 38406381
DOI: 10.36185/2532-1900-391 -
BioRxiv : the Preprint Server For... Feb 2024In biology, DNA is often tightly bent to small radii. Solely based on the groove asymmetry, a 30-year-old theoretical paper predicted that such bending should unwind...
In biology, DNA is often tightly bent to small radii. Solely based on the groove asymmetry, a 30-year-old theoretical paper predicted that such bending should unwind DNA, but this effect has not been directly experimentally quantified so far. We developed a ligation-based assay with nicked DNA circles of variable length, thereby decoupling the twist-dependent ligation efficiency from the large bending strain which dominates conventional circularization assays. We demonstrate that tightly bent DNA indeed unwinds to over 11 base pairs/turn, exactly as predicted. Our discovery requires reassessing the molecular mechanisms and energetics of all processes where DNA is tightly bent or relaxed again, including DNA packaging, gene regulation and expression.
PubMed: 38405957
DOI: 10.1101/2024.02.14.579968 -
Genes Feb 2024Congenital heart defects (CHDs) have had an increasing prevalence over the last decades, being one of the most common congenital defects. Their etiopathogenesis is...
Congenital heart defects (CHDs) have had an increasing prevalence over the last decades, being one of the most common congenital defects. Their etiopathogenesis is multifactorial in origin. About 10-15% of all CHD can be attributed to copy number variations (CNVs), a type of submicroscopic structural genetic alterations. The aim of this study was to evaluate the involvement of CNVs in the development of congenital heart defects. We performed a cohort study investigating the presence of CNVs in the 22q11.2 region and , , , , and genes in patients with syndromic and isolated CHDs. A total of 56 patients were included in the study, half of them (28 subjects) being classified as syndromic. The most common heart defect in our study population was ventricular septal defect (VSD) at 39.28%. There were no statistically significant differences between the two groups in terms of CHD-type distribution, demographical, and clinical features, with the exceptions of birth length, weight, and length at the time of blood sampling, that were significantly lower in the syndromic group. Through multiplex ligation-dependent probe amplification (MLPA) analysis, we found two heterozygous deletions in the 22q11.2 region, both in patients from the syndromic group. No CNVs involving , , , , and genes were identified in our study. We conclude that the MLPA assay may be used as a first genetic test in patients with syndromic CHD and that the 22q11.2 region may be included in the panels used for screening these patients.
Topics: Child; Humans; DNA Copy Number Variations; Pilot Projects; Multiplex Polymerase Chain Reaction; Cohort Studies; Romania; Heart Defects, Congenital
PubMed: 38397197
DOI: 10.3390/genes15020207 -
Plant Biotechnology Journal Jul 2024Advancement of DNA-synthesis technologies has greatly facilitated the development of synthetic biology tools. However, high-complexity DNA sequences containing tandems...
Advancement of DNA-synthesis technologies has greatly facilitated the development of synthetic biology tools. However, high-complexity DNA sequences containing tandems of short repeats are still notoriously difficult to produce synthetically, with commercial DNA synthesis companies usually rejecting orders that exceed specific sequence complexity thresholds. To overcome this limitation, we developed a simple, single-tube reaction method that enables the generation of DNA sequences containing multiple repetitive elements. Our strategy involves commercial synthesis and PCR amplification of padded sequences that contain the repeats of interest, along with random intervening sequence stuffers that include type IIS restriction enzyme sites. GoldenBraid molecular cloning technology is then employed to remove the stuffers, rejoin the repeats together in a predefined order, and subclone the tandem(s) in a vector using a single-tube digestion-ligation reaction. In our hands, this new approach is much simpler, more versatile and efficient than previously developed solutions to this problem. As a proof of concept, two different phytohormone-responsive, synthetic, repetitive proximal promoters were generated and tested in planta in the context of transcriptional reporters. Analysis of transgenic lines carrying the synthetic ethylene-responsive promoter 10x2EBS-S10 fused to the GUS reporter gene uncovered several developmentally regulated ethylene response maxima, indicating the utility of this reporter for monitoring the involvement of ethylene in a variety of physiologically relevant processes. These encouraging results suggest that this reporter system can be leveraged to investigate the ethylene response to biotic and abiotic factors with high spatial and temporal resolution.
Topics: Promoter Regions, Genetic; Plant Growth Regulators; Synthetic Biology; Plants, Genetically Modified; Cloning, Molecular; Gene Expression Regulation, Plant
PubMed: 38379432
DOI: 10.1111/pbi.14313