OBJECTIVETo identify the mutation of Cu/Zn superoxide dismutase(SOD1) gene in an amyotrophic lateral sclerosis (ALS) family with unique phenotype.

METHODSFive exons of SOD1 gene were amplified by PCR. The differences of these products were analyzed by PCR-single strand conformation polymorphism and visualized by silver staining.

RESULTSAbnormal bands were found in exons 2 and 5 of SOD1 gene in several familial members. DNA sequence analysis verified that a base pair insertion occurred in the codon area of exon 2 and in the intron area of exon 5. And the insertion mutation of exon 2 led to a frameshift mutation and premature stop. It is a new type of SOD1 mutation which may be associated with familial amyotrophic lateral sclerosis.

CONCLUSIONInsertion mutation of exon 2 may be responsible for the disease of an ALS family in Chongqing.

Adult ; Amyotrophic Lateral Sclerosis ; genetics ; Humans ; Mutation ; Polymorphism, Single-Stranded Conformational ; Superoxide Dismutase ; genetics ; Superoxide Dismutase-1

BACKGROUNDRNA interference (RNAi) is a potential cure for amyotrophic lateral sclerosis (ALS) caused by dominant, gain-of-function superoxide dismutase 1 (SOD1) mutations. The success of such therapy relies on the functional small interfering RNAs (siRNAs) that can effectively deliver RNAi. This study aimed to design the functional siRNAs targeting ALS-associated mutant alleles.

METHODSA modified dual luciferase system containing human SOD1 mRNA target was established to quantify siRNA efficacy. Coupled with validated siRNAs identified in the literature, we analyzed the rationale of siRNA design and subsequently developed an asymmetry rule-based strategy for designing siRNA. We then further tested the effectiveness of this design strategy in converting a naturally symmetric siRNA into functional siRNAs with favorable asymmetry for gene silencing of SOD1 alleles.

RESULTSThe efficacies of siRNAs could vary tremendously by one base-pair position change. Functional siRNAs could target the whole span of SOD1 mRNA coding sequence as well as non-coding region. While there is no distinguishable pattern of the distribution of nucleobases in these validated siRNAs, the high percent of GC count at the last two positions of siRNAs (P18 and P19) indicated a strong effect of asymmetry rule. Introducing a mismatch at position 1 of the 5' of antisense strand of siRNA successfully converted the inactive siRNA into functional siRNAs that silence SOD1 with desired efficacy.

CONCLUSIONSAsymmetry rule-based strategy that incorporates a mismatch into siRNA most consistently enhances RNAi efficacy and guarantees producing functional siRNAs that successfully silence ALS-associated SOD1 mutant alleles regardless target positions. This strategy could also be useful to design siRNAs for silencing other disease-associated dominant, gain-of-function mutant genes.

Amyotrophic Lateral Sclerosis ; genetics ; Cell Line ; Humans ; RNA Interference ; physiology ; RNA, Small Interfering ; genetics ; physiology ; Superoxide Dismutase ; genetics ; Superoxide Dismutase-1

OBJECTIVE: To explore the genetic basis for a Chinese patient with amyotrophic lateral sclerosis (ALS). METHODS: Peripheral blood samples were collected from the patient and his parents for the extraction of genomic DNA. Genetic variant was identified by whole exome sequencing. Candidate variant was verified by Sanger sequencing of his parents and healthy controls. RESULTS: The patient was found to harbor a heterozygous c.420C>G (p.Asn140Lys) variant of the SOD1 gene. The same variant was not detected in his parents and 100 healthy controls. The variant has not been included in HGMD, dbSNP and other databases. CONCLUSION The c.420C>G variant of the SOD1 gene may underlie the ALS in this patient. Above finding has enriched the spectrum of SOD1 gene variants.
Amyotrophic Lateral Sclerosis/genetics* ; China ; Heterozygote ; Humans ; Superoxide Dismutase-1/genetics* ; Whole Exome Sequencing

Human manganese superoxide dismutase (hMn-SOD) cDNA was amplified by RT-PCR from total RNA of human liver cell (L02), and cloned into yeast expression vector pPIC9K containing AOX1 promoter and the alpha-factor signal peptide sequence. The resultant pPIC9K-MnSOD was transformed to P. pastoris GS115, screened for Mut+ carrying multiple copies of hMn-SOD. The positive transformants were fermented in flasks and induced by 0.5% methanol. After 4 days of methanol induction, the expressed hMn-SOD was up to 32% of the total proteins in the supernatant by SDS-PAGE with specific activity of 247.7 u/mg.
Cloning, Molecular ; DNA, Complementary ; biosynthesis ; genetics ; Humans ; Pichia ; genetics ; metabolism ; Recombinant Proteins ; biosynthesis ; genetics ; Superoxide Dismutase ; biosynthesis ; genetics

BACKGROUND: Investigations of the pathogenic mechanisms in motor neurons (MNs) derived from amyotrophic lateral sclerosis (ALS) disease-specific induced pluripotent stem (iPS) cell lines could improve understanding of the issues affecting MNs. Therefore, in this study we explored mutant superoxide dismutase 1 (SOD1) protein expression in MNs derived from the iPS cell lines of ALS patients carrying different SOD1 mutations. METHODS: We generated induced pluripotent stem cell (iPSC) lines from two familial ALS (FALS) patients with SOD1-V14M and SOD1-C111Y mutations, and then differentiated them into MNs. We investigated levels of the SOD1 protein in iPSCs and MNs, the intracellular Ca2+ levels in MNs, and the lactate dehydrogenase (LDH) activity in the process of differentiation into the MNs derived from the controls and ALS patients' iPSCs. RESULTS: The iPSCs from the two FALS patients were capable of differentiation into MNs carrying different SOD1 mutations and differentially expressed MN markers. We detected high SOD1 protein expression and high intracellular calcium levels in both the MN and iPSCs that were derived from the two SOD1 mutant patients. However, at no time did we observe stronger LDH activity in the patient lines compared with the control lines. CONCLUSIONS MNs derived from patient-specific iPSC lines can recapitulate key aspects of ALS pathogenesis, providing a cell-based disease model to further elucidate disease pathogenesis and explore gene repair coupled with cell-replacement therapy. Incremental mutant expressions of SOD1 in MNs may have disrupted MN function, either causing or contributing to the intracellular calcium disturbances, which could lead to the occurrence and development of the disease.
Amyotrophic Lateral Sclerosis/genetics* ; Humans ; Induced Pluripotent Stem Cells ; Motor Neurons ; Mutation/genetics* ; Superoxide Dismutase-1/genetics*

BACKGROUNDSleep/wake disturbances in patients with amyotrophic lateral sclerosis (ALS) are well-documented, however, no animal or mechanistic studies on these disturbances exist. Orexin is a crucial neurotransmitter in promoting wakefulness in sleep/wake regulation, and may play an important role in sleep disturbances in ALS. In this study, we used SOD1-G93A transgenic mice as an ALS mouse model to investigate the sleep/wake disturbances and their possible mechanisms in ALS.

METHODSElectroencephalogram/electromyogram recordings were performed in SOD1-G93A transgenic mice and their littermate control mice at the ages of 90 and 120 days, and the samples obtained from these groups were subjected to quantitative reverse transcriptase-polymerase chain reaction, western blotting, and enzyme-linked immunosorbent assay.

RESULTSFor the first time in SOD1-G93A transgenic mice, we observed significantly increased wakefulness, reduced sleep time, and up-regulated orexins (prepro-orexin, orexin A and B) at both 90 and 120 days. Correlation analysis confirmed moderate to high correlations between sleep/wake time (total sleep time, wakefulness time, rapid eye movement [REM] sleep time, non-REM sleep time, and deep sleep time) and increase in orexins (prepro-orexin, orexin A and B).

CONCLUSIONSleep/wake disturbances occur before disease onset in this ALS mouse model. Increased orexins may promote wakefulness and result in these disturbances before and after disease onset, thus making them potential therapeutic targets for amelioration of sleep disturbances in ALS. Further studies are required to elucidate the underlying mechanisms in the future.

Amyotrophic Lateral Sclerosis ; genetics ; metabolism ; Animals ; Female ; Intracellular Signaling Peptides and Proteins ; genetics ; metabolism ; Male ; Mice ; Mice, Transgenic ; Neuropeptides ; genetics ; metabolism ; Orexins ; Reverse Transcriptase Polymerase Chain Reaction ; Sleep ; physiology ; Superoxide Dismutase ; genetics ; metabolism ; Superoxide Dismutase-1 ; Wakefulness ; physiology

OBJECTIVETo assess the association of extracellular superoxide dismutase (EC-SOD) gene methylation with cerebral infarction.

METHODSEighty-three patients with cerebral infarction and 94 healthy controls were enrolled. Based on cerebral MR findings, the size of infarction, extent of intracranial atherosclerosis, the National Institutes of Health Stroke Scale (NIHSS) score, and Barthel index were calculated. Methylation-specific PCR was used to analyze the methylation status of the EC-SOD gene in peripheral blood samples and its correlation with cerebral infarction.

RESULTSThe rate of EC-SOD gene promoter region methylation of the cerebral infarction group was lower than that of the control group (30.1% vs. 53.2%, P < 0.05). Patients with larger area of cerebral infarction (>4 cm in diameter) showed a lower methylation rate than those with a smaller cerebral infarction (0 vs. 39.1%, P < 0.05). Based on their cerebral MRA, 57 patients were divided into none, mild, moderate, and severe cerebral arteriosclerosis groups. The rate of EC-SOD gene methylation of the four groups showed a downward trend (at 45.5%, 42.9%, 23.8%, and 14.3%, respectively), though no statistical significance was found (P > 0.05). For the cerebral infarction group, those with higher rate of methylation had lower NIHSS scores (P < 0.05) but insignificantly higher Barthel index (P > 0.05).

CONCLUSIONThe EC-SOD methylation frequency of case group was lower than the control group. The methylation status is associated with the size of cerebral infarction, degree of cerebral arteriosclerosis and severity of neurological impairment.

Aged ; Cerebral Infarction ; genetics ; DNA Methylation ; Extracellular Space ; enzymology ; Female ; Humans ; Male ; Middle Aged ; Superoxide Dismutase ; genetics

OBJECTIVETo observe the association between SNPs in SOD1 (rs1041740, rs2070424, rs10432782 and rs4998557) and noise-induced hearing loss in Chinese Han population.

METHODSA case-control study was used to study the effects of environmental risk factors on the susceptibility to noise induced hearing loss (NIHL) in 201 sensitive workers and 202 resistant workers. A questionnaire was designed to carry out an investigation, and an occupational health survey was used to identify the occupational risk factors. Genomic DNA was extracted from peripheral blood cells using standard procedures of Takara kit, and 5 ml blood was from each subject. SNPs were detected using standard procedures of TaqMan probe allele identification method.

RESULTSIn SOD1 gene, the A allele of rs2070424 was a protective factor of NIHL, compared with the G allele (OR = 0.67, 95%CI: 0.50 approximately 0.88). The risk with NIHL in subjects with AA genotype was significantly lower than that in subjects with GG genotype (OR = 0.48, 95%CI: 0.26 approximately 0.79). After adjusting the confusion factors, OR was 0.44 and 95%CI was 0.25 approximately 0.78.

CONCLUSIONIn Chinese Han population, the SNP of rs2070424 in SOD1 may be associated with the susceptibility to NIHL.

Adolescent ; Adult ; Alleles ; Asian Continental Ancestry Group ; genetics ; Case-Control Studies ; Female ; Genetic Predisposition to Disease ; Genotype ; Haplotypes ; Hearing Loss, Noise-Induced ; epidemiology ; genetics ; Humans ; Male ; Noise, Occupational ; Polymorphism, Single Nucleotide ; Risk Factors ; Superoxide Dismutase ; genetics ; Superoxide Dismutase-1 ; Young Adult

The effects of genetic factors on the noise-induced hearing loss (NIHL) are still unclear. In the present study, eight single-nucleotide polymorphisms (SNPs) included rs1227049 and rs3802711 (CDH23), rs1695 (GSTP1), rs137852540 (GJB2), rs2289274 (PMCA2), rs4880 (SOD2), rs7943316, and rs769214 within CAT that might associated with NIHL were further validated in Chinese workers. The results showed that the carriers of the T allele (AT+TT) of rs7943316 and A allele (GA+AA) of rs769214, were significantly associated with an increased risk of NIHL compared to those with AA genotype (P<0.05) and GG genotype (P<0.05). Moreover, a significant three-locus model (P=0.0107) involving rs2016520, rs9794, and rs1805192 were observed that might associated with NIHL, with 53.95% of testing accuracy. Thus, our present study provided the evidence that GJB2, SOD2, and CAT genes might account for the NIHL development in independently and/or in an interactive manner.
Asian Continental Ancestry Group ; genetics ; Case-Control Studies ; Catalase ; genetics ; China ; Connexin 26 ; Connexins ; genetics ; Genetic Predisposition to Disease ; Hearing Loss, Noise-Induced ; genetics ; Humans ; Male ; Superoxide Dismutase ; genetics

Asian Continental Ancestry Group ; Case-Control Studies ; Humans ; Nitric Oxide Synthase Type III ; genetics ; Osteoarthritis ; genetics ; pathology ; PPAR gamma ; genetics ; Polymorphism, Genetic ; genetics ; Software ; Superoxide Dismutase ; genetics