Lysosomal dysfunction is a common pathological feature of neurodegenerative diseases. GTP-binding protein type A1 (GBA1) encodes beta-glucocerebrosidase 1 (GCase 1), a lysosomal hydrolase. Homozygous mutations in GBA1 cause Gaucher disease, the most common lysosomal storage disease, while heterozygous mutations are strong risk factors for Parkinson's disease. However, whether loss of GCase 1 activity is sufficient for lysosomal dysfunction has not been clearly determined. Here, we generated human neuroblastoma cell lines with nonsense mutations in the GBA1 gene using zinc-finger nucleases. Depending on the site of mutation, GCase 1 activity was lost or maintained. The cell line with GCase 1 deficiency showed indications of lysosomal dysfunction, such as accumulation of lysosomal substrates, reduced dextran degradation and accumulation of enlarged vacuolar structures. In contrast, the cell line with C-terminal truncation of GCase 1 but with intact GCase 1 activity showed normal lysosomal function. When alpha-synuclein was overexpressed, accumulation and secretion of insoluble aggregates increased in cells with GCase 1 deficiency but did not change in mutant cells with normal GCase 1 activity. These results demonstrate that loss of GCase 1 activity is sufficient to cause lysosomal dysfunction and accumulation of alpha-synuclein aggregates.
Cell Line ; Enzyme Activation/genetics ; Gene Knockout Techniques ; Gene Order ; Genetic Loci ; Glucosylceramidase/genetics/*metabolism ; Humans ; Lysosomes/*metabolism ; Mutation ; *Protein Aggregation, Pathological/genetics ; Protein Binding ; Zinc Fingers ; alpha-Synuclein/chemistry/*metabolism

Mitochondrial genomes have been extensively studied for phylogenetic purposes and to investigate intra- and interspecific genetic variations. In recent years, numerous groups have undertaken sequencing of platyhelminth mitochondrial genomes. Haplorchis taichui (family Heterophyidae) is a trematode that infects humans and animals mainly in Asia, including the Mekong River basin. We sequenced and determined the organization of the complete mitochondrial genome of H. taichui. The mitochondrial genome is 15,130 bp long, containing 12 protein-coding genes, 2 ribosomal RNAs (rRNAs, a small and a large subunit), and 22 transfer RNAs (tRNAs). Like other trematodes, it does not encode the atp8 gene. All genes are transcribed from the same strand. The ATG initiation codon is used for 9 protein-coding genes, and GTG for the remaining 3 (nad1, nad4, and nad5). The mitochondrial genome of H. taichui has a single long non-coding region between trnE and trnG. H. taichui has evolved as being more closely related to Opisthorchiidae than other trematode groups with maximal support in the phylogenetic analysis. Our results could provide a resource for the comparative mitochondrial genome analysis of trematodes, and may yield genetic markers for molecular epidemiological investigations into intestinal flukes.
Animals ; Asia ; Codon, Initiator ; DNA, Mitochondrial/chemistry/genetics ; Gene Order ; Genes, Helminth ; *Genome, Mitochondrial ; Heterophyidae/*genetics/isolation & purification ; Humans ; Molecular Sequence Data ; Sequence Analysis, DNA

OBJECTIVETo assess the association between SIRT1 gene polymorphisms and the longevity phenomena in Yongfu region of Guangxi. In this case-control study, 500 individuals from Yongfu region of Guangxi were recruited. The subjects were divided into a longevity group (n=223, average age=93.17 U+00B1 3.08 yr) and a healthy control group (n=277, average age=46.92 U+00B1 17.12 yr). Polymerase chain reaction-high resolution melting curve (PCR-HRM) and DNA sequencing were used to determine the allelic and genotypic frequencies of rs3758391, rs3740051, rs2273773, rs4746720 and rs10997870 polymorphisms of SIRT1 gene in the two groups. The association between above polymorphisms and longevity was assessed.

RESULTSIn the longevity group, CT genotype of the rs4746720 locus was significantly more common than CC and TT genotypes (P=0.000, OR=2.098, 95%CI:1.412-4.117). However, no significant difference was found in the allelic and genotypic frequencies of rs3758391, rs3740051 and rs2273773 between the two groups.

CONCLUSIONThere is an association between rs4746720 of SIRT1 gene and longevity in Yongfu region of Guangxi.

Adult ; Aged ; Aged, 80 and over ; Alleles ; Asian Continental Ancestry Group ; genetics ; Base Sequence ; Case-Control Studies ; China ; Female ; Gene Frequency ; Gene Order ; Genetic Association Studies ; Genotype ; Humans ; Longevity ; genetics ; Male ; Middle Aged ; Polymorphism, Single Nucleotide ; Sirtuin 1 ; genetics ; Young Adult

OBJECTIVETo develop a method for elucidating genetic basis of 21-hydroxylase deficiency.

METHODSSanger sequencing of entire 21-hydroxylase coding gene CYP21A2 was carried out to detect point mutations, and multiplex ligation-dependent probe amplification (MLPA) and locus-specific PCR/enzyme restriction method were used to detect large deletions and conversion mutations.

RESULTSNine children were analyzed. Point mutations of the CYP21A2 gene have been identified as: IVS2 13A/C>G (9 alleles), p.Arg356Trp (1 allele), Cluster E6 (1 allele), p.Gln318X (1 allele), and Prom conv (1 allele). While the former 4 mutations are pathogenic, the role of Prom conv mutation in the pathogenesis was uncertain. Three cases had entire CYP21A2 gene deletions (3 alleles), three had CYP21A1P/CYP21A2 chimeric mutations (3 alleles). The genotypes of all patients were determined. And all of the mutations were inherited from parents.

CONCLUSIONA rational method for detecting point mutations and large deletions/conversions of CYP21A2 gene has been established.

Adrenal Hyperplasia, Congenital ; diagnosis ; genetics ; Alleles ; Base Sequence ; Child ; Child, Preschool ; Female ; Gene Order ; Genotype ; Humans ; Infant ; Male ; Multiplex Polymerase Chain Reaction ; Steroid 21-Hydroxylase ; genetics

The C57BL/6J-fe/fe mouse is a coat color mutant. The coat color of the homozygote mouse becomes progressively lighter with advancing age. The faded gene (fe) of C57BL/6J-fe/fe was mapped in a 2.0 cM distal to D10mit191 by our group. To make a high-resolution map, we used the Korean wild mouse (KWHM) for a backcross panel, which was captured in 1995 and has been maintained as an inbred line by our laboratory. In the inter-specific backcross panel (N=400), the fe gene was mapped to 1.0 cM distal to D10mit156. The gene order was defined: centromere -D10mit3/85 (1.3+/-0.6 cM)-D10mit155 (1.3+/-0.6 cM)-D10mit191 (2.0+/-0.7 cM)-D10mit156 (1.0+/-0.5 cM)-fe-D10mit193 (1.3+/-0.6 cM)-D10mit54 (1.0+/-0.5 cM)-D10mit44 (8.5+/-1.4 cM)-D10mit42 (10.0+/-1.5 cM). The measured distance between D10mit191 and D10mit 44 differed in both inter-specific (DBA/2) and intra-specific (KWHM) backcross panels (14.2 vs 13.8 cM). Taken together, our high-resolution linkage map of the fe locus from an intra-specific backcross panel will provide a good entry point to isolate the fe gene.
Animals ; Centromere ; Chromosomes, Human, Pair 10 ; Gene Order ; Hair Color ; Homozygote ; Mice

OBJECTIVETo study the function of the chalcone synthese gene introns in Scutellaria baicalensis, and clarify preliminarily their role in abiotic stress.

METHODThe CHS introns with specific primers were cloned and bioinformatic method was applied to predict the cis-elements in the intron of CHS. The introns were subcloned into binary vector, pCAMBIA-1301 before being transferred to tobacco. Then the activity of GUS of the transgenic tobacco seeds was analyzed.

RESULTSeven cis-elements were found in the introns. Under the dark and high temperature GUS expression rose at the first (3 h), but then declined (9 h). ABA and MeJA regulated insignificantly the GUS activity in normal temperature; treatment of 10% PEG induced GUS expression.

CONCLUSIONCHS introns could be play a role in the regulation of S. baicalensis phenylpropanoid biosynthetic pathway.

Acyltransferases ; genetics ; Base Sequence ; Gene Expression Regulation, Plant ; drug effects ; Gene Order ; Genetic Vectors ; Introns ; Molecular Sequence Data ; Plants, Genetically Modified ; genetics ; metabolism ; Polyethylene Glycols ; pharmacology ; Regulatory Sequences, Nucleic Acid ; genetics ; Scutellaria baicalensis ; enzymology ; genetics ; Sequence Alignment ; Tobacco ; genetics ; metabolism

OBJECTIVETo investigate the type and frequency of gene conversion from SMN1 to SMN2 in Chinese patients affected with spinal muscular atrophy (SMA), and to explore the relationship between gene conversion and clinical phenotype.

METHODSNon-homozygous deletion of SMN1 gene exon 8 was screened among 417 patients with SMN1 exon 7 homozygous deletions. To analyze and verify the types of gene conversion, genomic DNA sequencing, multiplex ligation-dependent probe amplification (MLPA), and gene subcloning and sequencing were carried out.

RESULTSThirty-one patients (7.4% of all) with non-homozygous deletions of SMN1 exon 8 were detected. Through series of experiments, the fusion genes SMN1/SMN2 in all cases were delineated. Five types of gene conversions were identified, which included SMN2-I7b/SMN1 E8, SMN2-I7a/SMN1 I7b, SMN2-E7/SMN1 I7a, SMN1 I6/SMN2 E7/SMN1 I7a and SMN2-E7/SMN1 I7a/SMN2 I7b. Such conversions were found in the type I-III patients. For 10 patients with type I-III SMA and 3 copies of SMN2 gene produced by conversion, the average survival age was 5 year and 4 months.

CONCLUSIONPartial conversions of SMN1 gene have been found among Chinese SMA patients. The type of conversion and frequency seem to be different from those of other races. Gene conversion to some extent may impact on survival time and rate of SMA patients, especially type I SMA.

Base Sequence ; Child ; Child, Preschool ; Exons ; Female ; Gene Conversion ; Gene Order ; Homozygote ; Humans ; Infant ; Male ; Muscular Atrophy, Spinal ; genetics ; Phenotype ; Sequence Analysis, DNA ; Survival of Motor Neuron 1 Protein ; genetics ; Survival of Motor Neuron 2 Protein ; genetics

OBJECTIVEMutation screening was performed in a pedigree of Glanzmann's thrombasthenia (GT) and prenatal diagnosis was performed.

METHODSIn this study, reverse transcription-PCR-sequencing and PCR-sequencing, as well as restriction fragment length polymorphism(RFLP) and A/T-cloned-sequencing, were used to screen the ITGA2B and ITGB3 mutation in a pedigree with Glanzmann's thrombasthenia in the RNA and DNA level. Prenatal diagnosis was performed for this pedigree.

RESULTSDeletion of 99 bps was found in the cDNA of the patient in the pedigree, leading to deletion of 33 codons (from codon 160 to 192). After genomic analysis, the patient was found to be a compound heterozygote of c.374C to G mutation and intron 4(IVS-4) + 5 G to C mutation. The two mutations were inherited from the parents. IVS-4 + 5 G to C mutation was a point mutation in the splice site, while c.374C to G mutation was out of the splice site. But both of them resulted in the same splice pattern in RNA. The two mutations were novel mutations which have not been reported in Human Gene Mutation Database (HGMD) and the mutation data base of Glanzmann's thrombasthenia. The results of ITGB3 gene screening is normal in the proband and his parents.

CONCLUSIONTwo novel mutation, c.374C to G and IVS-4 + 5 G to C were found in this study, which might be the cause of GT in the pedigree.

Base Sequence ; Child, Preschool ; Female ; Gene Order ; Genetic Testing ; Genotype ; Humans ; Male ; Molecular Sequence Data ; Mutation ; genetics ; Pedigree ; Platelet Glycoprotein GPIIb-IIIa Complex ; chemistry ; genetics ; Pregnancy ; Prenatal Diagnosis ; Protein Conformation ; Thrombasthenia ; diagnosis ; genetics

The faded mouse is a coat color mutant that shows faded coat color and age-related loss of pigmentation. This mutation is transmitted by an autosomal recessive gene with 100% penetrance. In the present study, we carried out linkage analysis of the faded (fe) gene using intra-specific backcross panels. Affected faded mice were carefully confirmed by their faded coat color at about 4 weeks of age. In the intra-specific backcross between faded and CBA mice (n=198), the fe gene was mapped to a region 2.1 cM distal to D10mit191. Therefore, the gene order was defined as follows: centromere-D10mit51 (12.4+/-2.4 cM)-D10mit191 (2.1+/-1.0 cM)-fe-D10mit44 (13.3+/-2.4 cM)-D10mit42 (14.4+/-2.5 cM). This linkage map of the fe locus will provide a good entry point to isolate the fe gene. Since the faded mouse has pigmentary abnormalities, this mutant may be a useful model for studies of pigmentary abnormalities in humans.
Animals ; Chromosomes, Human, Pair 10 ; Gene Order ; Genes, Recessive ; Humans ; Mice ; Mice, Inbred CBA ; Penetrance ; Pigmentation

The study aimed to construct the amplicon vector of HSV-1 strain HF and explore its universal package function between different serotypes of HSV. OriS and pac elements were obtained by enzyme digestion from the Plasmid BAC-HSV-1 strain HF and sequenced. With red fluorescence (DsRed) as a reporter gene, the amplicon vector of HSV-1 strain HF was constructed based on pSilencer2.0-U6. The amplicon vector was transfected into Vero cells by lipofectamine 2000, then packaged by HSV-1 strain HF and HSV-2 strain HG52 as helper virus separately. The supernatant was collected after cytopathic effect. Red fluorescence was observed in Vero cells reinfected by the supernatant. In this study,the amplicon vector of HSV-1 strain HF was successfully constructed and it could be packaged by HSV-1 strain HF and HSV-2 strainHG52.
Animals ; Base Sequence ; Cercopithecus aethiops ; Gene Order ; Genes, Viral ; genetics ; Genetic Vectors ; genetics ; Herpesvirus 1, Human ; classification ; genetics ; Herpesvirus 2, Human ; genetics ; Molecular Sequence Data ; Replication Origin ; genetics ; Serotyping ; Vero Cells