AIMTo develop a simple, fast and inexpensive approach as well as an instrument for detection of gene mutation.

METHODSPyrosequencing based on bioluminometry assay was employed to detect gene mutation. Pyrosequencing is a method of sequencing by synthesis step-by-step using four enzymes, DNA-polymerase, ATP sulfurylase, luciferase and apyrase. The signal was produced by detecting pyrophosphate released during a dNTP incorporation. For mutation detection, a DNA fragment was amplified by PCR at first, followed by a single-stranded DNA preparation. In the second step, a short primer was annealed to the position just before the mutation point. Finally, specific dNTPs were added in terms of the template sequence. The mutation species can be readily determined by the sequence.

RESULTSA new instrument was developed for gene mutation detection by pyrosequencing. To iteratively inject small amount of each dNTP for the sequencing reaction, capillaries were used to connect dNTP reservoirs and the reaction chamber. Each dNTP was delivered by adding a gas pressure on the top of a corresponding dNTP reservoir, by which 0.2 microL of dNTP can be exactly added each time. It was theoretically proved that undesired liquid seep through the capillary did not affect the sequencing reactions in pyrosequencing. In addition, the three possible variants (wildtype, mutant and heterozygote) of a mutant point Cys275Ser in P53 gene exon 8 were determined by pyrosequencing using the instrument. A simple method was also described for rapidly distinguishing the type of a variant.

CONCLUSIONThe developed method is very simple, and the corresponding instrument is inexpensive and easy to operate, which can be used to detect many types of mutation.

Exons ; genetics ; Genes, p53 ; genetics ; Humans ; Luminescent Measurements ; Point Mutation

OBJECTIVE: To explore the characteristics of PAH gene variants among 113 phenylketonuria patients from Henan Province. METHODS: The 13 exons of the PAH gene were subjected to PCR amplification and direct sequencing. Large fragment deletion and duplication of the PAH gene were detected with a multiple ligation-dependent probe amplification (MLPA) assay. RESULTS: In total 195 point variants and 3 large fragment deletions were detected among the 226 alleles, with the detection rates being 86.28% and 1.33%, respectively. Variants of p.Arg243Gln (18.14%), p.Arg111X (6.19%), p.Arg53His (5.31%), EX6-96A>G (5.31%), p.Tyr356X (4.87%) and p.Val399Val (4.42%) were relatively common. Most of the variants were located in exons 7, 11, 3 and 6. Missense variations were most common. Four novel variations were detected, which included c.1016C>A (p.Ser339Tyr), c.1000T>C (p.Cys334Arg), c.1110G>T (p.Glu370Asp), and IVS6+1G>T. CONCLUSION The PAH gene variations in Henan Province have featured extensive allelic heterogeneity and variety.
China ; Exons ; Humans ; Mutation, Missense ; Phenylalanine Hydroxylase ; genetics ; Phenylketonurias ; genetics ; Point Mutation ; Sequence Deletion

To establish a rabbit model of proprotein convertase subtilisin/kexin type9 () point mutation with CRISPR/Cas9 gene editing technique. According to the PubMed gene protein data, the PCSK9 protein functional regions of human and rabbit were analyzed by Blast. The 386S (Ser) amino acid functional region of human gene was homologous to the 485S of rabbit gene. Three small guide RNAs and one single-stranded donor oligonucleotide were designed according to the 485S base substitution position and sequence analysis of rabbit gene. The synthetic small guide RNAs, Cas9 mRNA and single-stranded donor oligonucleotide were co-injected into the cytoplasm of rabbit fertilized eggs and the embryos were transferred into the pregnant rabbits. PCR, TA cloning and off-target analysis were performed on the F0 rabbits to identify whether the PCSK9 mutation was successful. Fifteen F0 rabbits were obtained. The sequencing results showed that one of them was PCSK9 point mutation homozygote and two of them were PCSK9 point mutation heterozygotes, and the mutation could be stably inherited. The rabbit model of PCSK9 point mutation was successfully constructed by CRISPR/Cas9 technique, which provides an animal model for exploring the molecular mechanism of impaired PCSK9 function and developing reliable and effective diagnosis and treatment measures.
Animals ; CRISPR-Cas Systems/genetics* ; Mutation ; Point Mutation ; Proprotein Convertase 9/metabolism* ; Rabbits

OBJECTIVETo investigate mutations in the D-loop region of mitochondrial DNA in ovarian tumors.

METHODSThe D-loop region of 25 epithelial ovarian tumors together with the adjacent normal tissues were amplified by PCR and sequenced.

RESULTSAmong the 25 ovarian tumors, 26 mutations were identified with the mutation rate of 32%. 19 mutations were detected in two cases of borderline carcinoma which was a special type of epithelial ovarian carcinoma. There were 6 microsatellite instabilities among the mutations and 11 new polymorphisms which were not reported previously in the GenBank.

CONCLUSIONSThe D-loop region of mitochondrial DNA is a highly polymorphoric and mutable region and the mutation rate is relatively high in patients with ovarian tumors.

Cystadenocarcinoma, Serous ; genetics ; DNA, Mitochondrial ; genetics ; Female ; Humans ; Ovarian Neoplasms ; genetics ; Point Mutation ; Polymorphism, Genetic

OBJECTIVETo study the transcriptional regulation of human delta globin gene with C-->T point mutation at -64 in its promoter.

METHODSHuman delta globin genes including wild CAAT box and mutant CAAT box (-64C-->T) were separately cloned into eukaryotic expression vector pcDNA3.1 (-)/Myc-His A which was cut out the strong promoter CMV, transfected MEL cells, and induced by DMSO to express. The transcriptional regulation of human delta globin gene was analysed using semi-quantitative RT-PCR.

RESULTSThe expression level of human delta globin gene with mutant CAAT box was 2.2-fold as high as that with wild CAAT box.

CONCLUSIONThe defective CAAT box of human delta globin gene promoter region may be one of the major reasons for its low expression level.

CCAAT-Enhancer-Binding Proteins ; genetics ; Globins ; genetics ; Humans ; Point Mutation ; Promoter Regions, Genetic ; genetics ; Transcription, Genetic

Mitochondria is the main place of biological oxidation and energy transform. Mitochondrial DNA encodes the complex of respiratory chain in mitochondria and its mutation can cause a series of human disease. Mitochondrial DNA mutation which observed in myelodysplastic syndrome (MDS) patients cause the MDS by the mechanism of iron metabolism disorder, gene instability and hemopoietic progenitor cell apoptosis. In this review the characteristics of mitochondrial DNA structure, the mitochondrial DNA mutation and the possible mechanism of mitochondrial DNA mutation in pathogenesis of MDS were summarized.
Anemia, Sideroblastic ; genetics ; DNA, Mitochondrial ; genetics ; Humans ; Myelodysplastic Syndromes ; complications ; genetics ; Point Mutation

Mutations in the parkin gene have recently been identified in familial and isolated patients with early-onset Parkinson disease (PD) and that subregions between exon 2 and 4 of the parkin gene are hot spots of deletive mutations. To study the distribution of deletions in the parkin gene among variant subset patients with PD in China, and to explore the role of parkin gene in the pathogenesis of PD, 63 patients were divided into early onset and later onset groups. Exons 1-12 were amplified by PCR, templated by the genomic DNA of patients, and then the deletion distribution detected by agarose electrophoresis. Four patients were found to be carrier of exon deletions in 63 patients with PD. The location of the deletion was on exon 2 (1 case), exon 3 (2 cases) and exon 4 (1 case). All patients were belong to the group of early onset PD. The results showed that parkin gene deletion on exon 2, exon 3 and exon 4 found in Chinese population contributes partly to early onset PD.
Exons/*genetics ; *Gene Deletion ; Parkinson Disease/*genetics ; Point Mutation ; Ubiquitin-Protein Ligases/*genetics

There are some human diseases associated with mitochondrial DNA genome defect. Now many studies think that: oxygen radical resulting from oxidative phosphorylation(OXPHOS) disorder caused by myocardium ischemia and the increased OXPHOS induction damage mitochondrial DNA. Chronic damage accumulations lead to mitochondrial DNA deletion or point mutation in the end which show mitochondrial DNA 5.0 kb or 7.4 kb deletion and point mutation at position C15452A in the cytochrome b gene; the conservative sequence mutation of tRNA gene such as A4300G, C4320T point mutation in the tRNA Ilegene, A3243G point mutation in the tRNA leu gene etc result in defective contractile proteins whose persistent and inefficient contraction may increase the myocardium's metabolic demands for ATP and leads to cardiac hypertrophy. In this article, we review the study on the association of mitochondrial DNA mutation with ischemic cardiomyopathy and hypertrophic cardiomyopathy.
Cardiomyopathy, Hypertrophic/genetics* ; DNA, Mitochondrial/genetics* ; Humans ; Myocardial Ischemia/genetics* ; Point Mutation

OBJECTIVEAlport syndrome (AS) is a progressive hereditary nephritis presented with hematuria and renal failure, frequently associated with sensorineural deafness and ocular lesions. So far, more than 300 gene mutations in AS have been identified which provides a better way to analyze the association between genotype and phenotype. It is hard to understand all the phenotype according to the gene mutations, because the structure and function changes of the relevant protein, alpha5(IV) chain, encoded by mutated COL4A5 gene are rare to know. This study aimed to detect the proteins structure encoded by COL4A5 gene with different missense mutations and to analyze the effect of gene mutations on the secondary structure of alpha5(IV) chain and structure-phenotype relations.

METHODSTwo X-linked AS patients with different missense mutations (g.3246G > T resulting in p.G1015V and g.3290G > A resulting in p.G1030S, respectively) diagnosed by clinical manifestations, family history and skin or renal biopsy examinations, as well as a control were included in this study. The fragments of cDNA with the two mutations, respectively, and that of corresponding cDNA from the control were expressed in E. coli. The secondary structure of the recombinant polypeptides were analyzed by using circular dichroism (CD) spectroscopy.

RESULTSCD spectra of the control exhibited a negative peak near 200 nm whereas that of the patient 1 exhibited a negative peak near 220 nm. Furthermore, the magnitude of the negative peak of patient 1 decreased from -9000 deg x cm2 x dmol(-1) to -4000 deg x cm2 x dmol(-1) as compared with that of the control. CD spectra of the patient 2 were slightly changed with the negative peak remaining near 220 nm but the magnitude increasing from -9000 deg x cm2 x dmol(-1) to -11000 deg x cm2 x dmol(-1) as compared with that of the control. In addition, the secondary structure of the control polypeptide was mainly composed of beta-sheet and random coil without alpha-helix, whereas that of the patient 1 presented 12.9% alpha-helix. Although the secondary structure of polypeptide of the patient 2 was also mainly composed of beta-sheet and random coil, the composition of beta-sheet reduced and random coil increased.

CONCLUSIONAlthough the glycine substitutions existed in the same domain of alpha5(IV) chain, the patient 1 with the severe AS phenotype and g.3246G > T mutation, and patient 2 with the mild AS phenotype and g.3290G > A mutation were revealed with different secondary structures of alpha5(IV) chain. Moreover, the secondary structure changes of alpha5(IV) chain were consistent with their corresponding phenotype severity.

Collagen Type IV ; genetics ; Humans ; Mutation, Missense ; Nephritis, Hereditary ; genetics ; Phenotype ; Point Mutation ; Protein Structure, Secondary ; genetics ; Spectrum Analysis

Cardiomyopathy, Dilated ; genetics ; DNA, Mitochondrial ; genetics ; Humans ; Hypertension ; genetics ; Mitochondrial Diseases ; genetics ; Mitochondrial Proton-Translocating ATPases ; genetics ; Point Mutation ; genetics