Nucleic acid aptasensor with magnetically induced self-assembly for the detection of EpCAM glycoprotein.

A "turn-on" aptasensor for label-free and cell-free EpCAM detection was constructed by employing magnetic α-Fe2O3/Fe3O4@Au nanocomposites as a matrix for signal amplification and double-stranded complex (SH-DNA/Apt probes) immobilization through Au-S binding. α-Fe2O3/Fe3O4@Au could be efficiently assembled into uniform and stable self-assembly films via magnetic-induced self-assembly technique on a magnetic glassy carbon electrode (MGCE). The effectiveness of the platform for EpCAM detection was confirmed through differential pulse voltammetry (DPV). Under optimized conditions, the platform exhibited excellent specificity for EpCAM, and a strong linear correlation was observed between the current and the logarithm of EpCAM protein concentration in the range 1 pg/mL-1000 pg/mL (R2 = 0.9964), with a limit of detection (LOD) of 0.27 pg/mL. Furthermore, the developed platform demonstrated good stability during a 14-day storage test, with fluctuations remaining below 93.33% of the initial current value. Promising results were obtained when detecting EpCAM in spiked serum samples, suggesting its potential as a point-of-care (POC) testing.

Fabrication of magneticα-Fe2O3/Fe3O4heterostructure nanorods via the urea hydrolysis-calcination process and their biocompatibility with LO2and HepG2cells.

ß-FeOOH nanorods were prepared via the urea hydrolysis process with the average length of 289.1 nm and average diameter of 61.2 nm, while magneticα-Fe2O3/Fe3O4heterostructure nanorods were prepared via the urea calcination process withß-FeOOH nanorods as precursor, and the optimum conditions were the calcination temperature of 400 °C, the calcination time of 2 h, theß-FeOOH/urea mass ratio of 1:6. The average length, diameter, and the saturation magnetization of the heterostructure nanorods prepared under the optimum conditions were 328.8 nm, 63.4 nm and 42 emu·g-1, respectively. The Prussian blue test demonstrated that the heterostructure nanorods could be taken up by HepG2 cells, and cytotoxicity tests proved that the heterostructure nanorods had no significant effect on the viabilities of LO2 and HepG2 cells within 72 h in the range of 100-1600µg·ml-1. Therefore, magneticα-Fe2O3/Fe3O4heterostructure nanorods had better biocompatibility with LO2 and HepG2 cells.

Immobilization and property of penicillin G acylase on amino functionalized magnetic Ni0.3Mg0.4Zn0.3Fe2O4 nanoparticles prepared via the rapid combustion process.

Penicillin G acylase plays an important role in the biocatalytic process of semi-synthetic penicillin. In order to overcome the disadvantages of free enzymes and improve the catalytic performance of enzymes, it is a new method to immobilize enzymes on carrier materials. And magnetic materials have the characteristics of easy separation. In the present study, the Magnetic Ni0.3Mg0.4Zn0.3Fe2O4 nanoparticles were successfully prepared by a rapid-combustion method and calcined at 400°C for 2 h. The surface of the nanoparticles was modified with sodium silicate hydrate, and the PGA was covalently bound to the carrier particles through the cross-linking of glutaraldehyde. The results showed that the activity of immobilized PGA reached 7121.00 U/g. The optimum pH for immobilized PGA was 8 and the optimum temperature was 45°C, the immobilized PGA exhibited higher stability against changes in pH and temperature. The Michaelis-Menten constant (Km) values of the free and immobilized PGA were 0.00387 and 0.0101 mol/L and the maximum rate (Vmax) values were 0.387 and 0.129 µmol/min. Besides, the immobilized PGA revealed excellent cycling performance. The immobilization strategy presented PGA had the advantages of reuse, good stability, cost saving and had considerable practical significance for the commercial application of PGA.

Effect and mechanism of paclitaxel loaded on magnetic Fe3O4@mSiO2-NH2-FA nanocomposites to MCF-7 cells.

Magnetic Fe3O4 nanoparticles were prepared via a simple hydrothermal method and utilized to load paclitaxel. The average particle size of Fe3O4 nanoparticles was found to be 20.2 ± 3.0 nm, and the calculated saturation magnetization reached 129.38 emu/g, verifying superparamagnetism of nanomaterials. The specific surface area and pore volume were 84.756 m2/g and 0.265 cm3/g, respectively. Subsequently, Fe3O4@mSiO2 nanoparticles were successfully fabricated using the Fe3O4 nanoparticles as precursors with an average size of 27.81 nm. The relevant saturation magnetization, zeta potential, and specific surface area of Fe3O4@mSiO2-NH2-FA were respectively 76.3 emu/g, -14.1 mV, and 324.410 m2/g. The pore volume and average adsorption pore size were 0.369 cm3/g and 4.548 nm, respectively. Compared to free paclitaxel, the solubility and stability of nanoparticles loaded with paclitaxel were improved. The drug loading efficiency and drug load of the nanoformulation were 44.26 and 11.38%, respectively. The Fe3O4@mSiO2-NH2-FA nanocomposites were easy to construct with excellent active targeting performance, pH sensitivity, and sustained-release effect. The nanoformulation also showed good biocompatibility, where the cell viability remained at 73.8% when the concentration reached 1200 µg/mL. The nanoformulation induced cell death through apoptosis, as confirmed by AO/EB staining and flow cytometry. Western blotting results suggested that the nanoformulation could induce iron death by inhibiting Glutathione Peroxidase 4 (GPX4) activity or decreasing Ferritin Heavy Chain 1 (FTH1) expression. Subsequently, the expression of HIF-1α was upregulated owing to the accumulation of reactive oxygen species (ROS), thus affecting the expression of apoptosis-related proteins regulated by p53, inducing cell apoptosis.

Correlation Between Brain Structure Atrophy and Plasma Amyloid-ß and Phosphorylated Tau in Patients With Alzheimer's Disease and Amnestic Mild Cognitive Impairment Explored by Surface-Based Morphometry.

Objective: To investigate the changes in the cortical thickness of the region of interest (ROI) and plasma Aß40, Aß42, and phosphorylated Tau (P-Tau) concentrations in patients with Alzheimer's disease (AD) and amnestic mild cognitive impairment (aMCI) as the disease progressed with surface-based morphometry (SBM), to analyze the correlation between ROI cortical thickness and measured plasma indexes and neuropsychological scales, and to explore the clinical value of ROI cortical thickness combined with plasma Aß40, Aß42, and P-Tau in the early recognition and diagnosis of AD. Methods: This study enrolled 33 patients with AD, 48 patients with aMCI, and 33 healthy controls (normal control, NC). Concentration changes in plasma Aß42, Aß40, and P-Tau collected in each group were analyzed. Meanwhile, the whole brain T1 structure images (T1WI-3D-MPRAGE) of each group of patients were collected, and T1 image in AD-aMCI, AD-NC, and aMCI-NC group were analyzed and processed by SBM technology to obtain brain regions with statistical differences as clusters, and the cortical thickness of each cluster was extracted. Multivariate ordered logistic regression analysis was used to screen out the measured plasma indexes and the indexes with independent risk factors in the cortical thickness of each cluster. Three comparative receiver operating characteristic (ROC) curves of AD-aMCI, AD-NC, and aMCI-NC groups were plotted, respectively, to explore the diagnostic value of multi-factor combined prediction for cognitive impairment. The relationship between cortical thickness and plasma indexes, and between cortical thickness and Mini-Mental State Examination (MMSE) and Montreal Cognitive Assessment (MoCA) scores were clarified by Pearson correlation analysis. Results: Plasma Aß40, Aß42, and P-Tau proteins in the NC, aMCI, and AD groups increased with the progression of AD (P < 0.01); cortical thickness reductions in the AD-aMCI groups and AD-NC groups mainly involved the bilateral superior temporal gyrus, transverse temporal gyrus, superior marginal gyrus, insula, right entorhinal cortex, right fusiform gyrus, and cingulate gyrus. However, there were no statistical significances in cortical thickness reductions in the aMCI and NC groups. The cortical thickness of the ROI was negatively correlated with plasma Aß40, Aß42, and P-Tau concentrations (P < 0.05), and the cortical thickness of the ROI was positively correlated with MMSE and MoCA scores. Independent risk factors such as Aß40, Aß42, P-Tau, and AD-NC cluster 1R (right superior temporal gyrus, temporal pole, entorhinal cortex, transverse temporal gyrus, fusiform gyrus, superior marginal gyrus, middle temporal gyrus, and inferior temporal gyrus) were combined to plot ROC curves. The diagnostic efficiency of plasma indexes was higher than that of cortical thickness indexes, the diagnostic efficiency of ROC curves after the combination of cortical thickness and plasma indexes was higher than that of cortical thickness or plasma indexes alone. Conclusion: Plasma Aß40, Aß42, and P-Tau may be potential biomarkers for early prediction of AD. As the disease progressed, AD patients developed cortical atrophy characterized by atrophy of the medial temporal lobe. The combined prediction of these region and plasma Aß40, Aß42, and P-Tau had a higher diagnostic value than single-factor prediction for cognitive decline.

The c.323 G>C mutation in LORICRIN causes new-found late-onset autosomal dominant loricrin keratoderma in a Chinese Han Pedigree.

BACKGROUND: Loricrin keratoderma is a rare early-onset autosomal dominant skin disorder. At present, no clinical reports have been published on characteristics of progressive aggravation and late-onset. OBJECTIVES: To identified a new-found pedigree with c.323 G>C mutation leading to progressive aggravation and late-onset loricrin keratoderma. METHODS: Targeted next-generation sequencing of 267 genes associated with all skin abnormalities, sanger sequencing, and bioinformatics tools were used to identify the mutation in this new-found pedigree. Palm skin biopsy was used to observe the clinicopathological features of patient. Further, we constructed pcDNA3.1/V5-His-wild-LORICRIN, pcDNA3.1/V5-His-c.323G>C-LORICRIN, and pcDNA3.1/V5-His-730insG-LORICRIN vectors, nucleofected into HaCaT strain to observe the subcellular localization of loricrin by using the laser scanning confocal microscopy. RESULTS: The proband and his affected father carried a heterozygous c.323 G>C missense mutation (p.Gly108Ala) on LORICRIN. Bioinformatics analysis hinted that it had potential pathogenicity; the types of ligands, enzyme commission active sites, and the spatial structure of protein changed enormously. Laser scanning confocal microscopy showed that the signals from cells transfected with the pcDNA3.1/V5-His-730insG-LORICRIN vector were distributed mainly in the nucleus, whereas those from cells transfected with the pcDNA3.1/V5-His-c.323G>C-LORICRIN vector were mainly located in the cytoplasm. Wild type loricrin was distributed in the nucleus and cytoplasm homogeneously CONCLUSION: The heterozygous c.323G>C missense mutation on LORICRIN caused late-onset and progressive loricrin keratoderma in this large Chinese family. Our study revealed that a large number of loricrin gathered in the cytoplasm may disturb the normal proliferation and terminal differentiation of keratinocytes and lead to the late-onset loricrin keratoderma disease.

A novel electrochemical DNA biosensor for transgenic soybean detection based on triple signal amplification.

A novel electrochemical DNA biosensor was developed and MON89788 of soybean transgenic gene sequence was detected based on a strategy of rolling circle amplification (RCA) and gold nanoparticle cube (AuNPC)-labeled multiple probes. First, the mercapto-modified capture DNA was immobilized on the surface of the Fe3O4@Au magnetic nanoparticles via an Au-S bond, and the capture DNA was opened and complementarily hybridized with the target DNA to form a double-stranded DNA. In the 10 × reaction buffer, Exonuclease III (ExoIII) specifically recognized and sheared the double-stranded DNA to release the target DNA, which led to the next round of reaction. Afterward, AuNP cube-loaded ssDNA (AuNPC/DNA) was added with the rolling circle reaction with the help of Phi29 DNA polymerase and T4 ligase. Finally, [Ru(NH3)6]3+ was attracted directly by the anionic phosphate of ssDNA via electrostatic interaction. The determination was carried out by using chronocoulometry (CC), and the CC signal was recorded. The mass amount of DNA strands extended infinitely on the AuNPs cube and numerous [Ru(NH3)6]3+ were absorbed, thus the detected signal was highly amplified. The corresponding CC signal showed a good linear relationship with the logarithm of the target DNA concentration in the range of 1 × 10-16 to 1 × 10-7 mol L-1, with a detection limit of 4.5 × 10-17 mol L-1. Specific gene sequence of MON89788 in soybean samples was determined, and the recoveries ranged from 97.3% to 102.0%. This sensor is one of the most sensitive sensors for genetic sequence assessment at present. Moreover, it demonstrates good selectivity, stability, and reproducibility.

Protective effect of treatment with low-dose gliclazide in a model of middle cerebral artery occlusion and reperfusion in rats.

The aim of this study was to explore the expression of sulfonylurea receptor 1 (SUR1), the regulatory subunit of the NCCa-ATP channel, and to investigate the protective effects of gliclazide following middle cerebral artery occlusion (MCAO)/reperfusion in male Wistar rats. Adult rats underwent 2h of the left MCAO using the intraluminal thread technique before reperfusion. The core areas of the infarct at different reperfusion time points were examined for the mRNA level and protein expression of SUR1 using reverse transcription-polymerase chain reaction (RT-PCR) and western blotting respectively. Gliclazide was administered intravenously into the right jugular vein for 12h simultaneously with the reperfusion. The number of apoptotic cells was determined using the TUNEL assay. The neurological functional deficits were evaluated using Bederson׳s test, and the cerebral infarction volume was visualized with TTC staining. We found up-regulation of SUR1 mRNA and protein levels in ischemic infarct tissues after reperfusion following MCAO, and SUR1 mRNA and protein were maximally upregulated 8-12h after a 2-hour ischemia. The treatment with low-dose of gliclazide reduced the total number of TUNEL-positive cells, the neurological functional deficits and the brain infarct volume. These results suggest that the SUR1-regulated NCCa-ATP channel may be associated with MCAO/reperfusion injury and the infarct-reducing effects of intravenous treatment with gliclazide may be due, in part, to the blocked upregulation of SUR1 expression, the decreased infarct size and the reduced apoptosis in the ischemia-reperfusion brain.

Synthesis and biological evaluation of Combretastatin A-4 derivatives containing a 3'-O-substituted carbonic ether moiety as potential antitumor agents.

BACKGROUND: Combretastatin A-4 (CA-4), which is an excellent antineoplastic agent, was isolated from Combretum caffrum. To date, structural modification studies of CA-4 have focused predominantly on the construction of new therapeutic agents for drug discovery. As a part of our ongoing work towards the modification of natural products, we have focused on the 3'-O-substituent groups in the B-ring of CA-4 under the hypothesis that these novel derivatives will possess good bioactivities and behave as effective antiproliferative pro-drugs. RESULTS: A series of novel CA-4 derivatives, which contained a 3'-O-substituted carbonic ether moiety, were synthesized and evaluated for their antitumor activities against four tumor cell lines, including MDA-MB-231, MCF-7, K562 and A549 cells. These derivatives exhibited clear antitumor activities, and CA-4E, in particular, showed the highest bioactivity of all of the derivatives tested against all four tumor cell lines, with IC50 values in the range of 1 to 180 nM. Based on its high bioactivity, CA-4E was subsequently selected to investigate the antitumor mechanism of these synthetic compounds. The cell cycle results demonstrated that CA-4E induced time- and dose-dependent G2/M arrest in a similar manner to CA-4, although its effect was more powerful than that of CA-4, and the apoptosis data showed that CA-4E induced cellular apoptosis in a dose-dependent manner. CONCLUSIONS: The newly synthesized CA-4 derivatives exhibited good antitumor activities in vitro, with CA-4E, in particular, showing the highest bioactivity of all of the compounds tested. Furthermore, CA-4E induced time- and dose-dependent G2/M arrest and cellular apoptosis in a dose-dependent manner. Taken together, these results suggest that CA-4E should be subjected to further investigation as a potential anticancer drug candidate.

[Analysis of PHEX gene mutation in a hypophosphatasia pedigree].

OBJECTIVE: To screen potential mutations of PHEX gene in a family featuring hypophosphatemic rickets in order to confirm the molecular diagnosis and pathogenetic mechanism. METHODS: Genomic DNA was extracted from peripheral venous blood samples. DNA sequence of PHEX gene was derived from UCSC database, and primers for its coding region were designed with Primer premier 5.0. Potential mutations were detected with PCR amplification and DNA sequence analysis. RESUTLS: A mutation was identified in intron 6 of the PHEX gene in the proband and his mother. CONCLUSION: The c.732+1G>T mutation underlies the hypophosphatemic rickets in this family.

TARDBP gene mutations among Chinese patients with sporadic amyotrophic lateral sclerosis.

Recently, several TARDBP mutations have been identified in sporadic amyotrophic lateral sclerosis (SALS) patients among different ethnicities. Our study aims to analyze the clinical features and mutations in the TARDBP gene among Chinese patients with SALS. One hundred sixty-five patients were studied. The mean age of onset was 50.8±12.0 years. The mean diagnostic delay was 18.8±17.1 months. A novel missense mutation (p.N378S) and a novel silent change (p.A321A) were detected in 2 male patients, respectively. A new variant of c.1098C>G in exon 6 and 2 reported variants, g.IVS1+85C>T in intron 1 and c.57A>G in exon 2, were found. The frequency of the "G" variant of c.57A>G in exon 2 and the "G" variant of c.1098C>G in exon 6 were significantly lower in the patient group than in the control (p=0.001 and p=0.024, respectively). Our findings provide first evidence that the frequency of TARDBP gene mutations is rare among Chinese SALS patients (0.61%). Several polymorphisms may influence susceptibility to amyotrophic lateral sclerosis.

Identification of novel mutations in Chinese Hans with autosomal dominant polycystic kidney disease.

BACKGROUND: Autosomal dominant polycystic kidney disease (ADPKD) is the most common inherited renal disease with an incidence of 1 in 400 to 1000. The disease is genetically heterogeneous, with two genes identified: PKD1 (16p13.3) and PKD2 (4q21). Molecular diagnosis of the disease in at-risk individuals is complicated due to the structural complexity of PKD1 gene and the high diversity of the mutations. This study is the first systematic ADPKD mutation analysis of both PKD1 and PKD2 genes in Chinese patients using denaturing high-performance liquid chromatography (DHPLC). METHODS: Both PKD1 and PKD2 genes were mutation screened in each proband from 65 families using DHPLC followed by DNA sequencing. Novel variations found in the probands were checked in their family members available and 100 unrelated normal controls. Then the pathogenic potential of the variations of unknown significance was examined by evolutionary comparison, effects of amino acid substitutions on protein structure, and effects of splice site alterations using online mutation prediction resources. RESULTS: A total of 92 variations were identified, including 27 reported previously. Definitely pathogenic mutations (ten frameshift, ten nonsense, two splicing defects and one duplication) were identified in 28 families, and probably pathogenic mutations were found in an additional six families, giving a total detection level of 52.3% (34/65). About 69% (20/29) of the mutations are first reported with a recurrent mutation rate of 31%. CONCLUSIONS: Mutation study of PKD1 and PKD2 genes in Chinese Hans with ADPKD may contribute to a better understanding of the genetic diversity between different ethnic groups and enrich the mutation database. Besides, evaluating the pathogenic potential of novel variations should also facilitate the clinical diagnosis and genetic counseling of the disease.

Evidence for a predisposing background for CAG expansion leading to HTT mutation in a Chinese population.

OBJECTIVE: To investigate the predisposing background for the instability of CAG expansions of the HTT gene in a Chinese population. METHODS: Genotyping and haplotyping of CAG and CCG repeats of the HTT gene were carried out in 32 unrelated HD patients and 95 non-HD control individuals of Han origin, using capillary electrophoresis and DNA sequencing. The frequencies of different CCG repeats were compared between mutant and wild-type HTT genes. In controls, the comparison of the mean CAG repeat size was performed among different CCG repeats. RESULTS: A total of five alleles of CCG repeats were distinguished, in which four were present in HD chromosomes. In the CCG alleles, (CCG)10 showed a higher frequency in mutant HTT genes relative to wild-type ones, and the highest mean CAG repeat size was observed in the (CCG)10 background. Additionally, a haplotype of (CAG)32-(CCG)10 was found in the control group. CONCLUSION: Our findings indicate that HTT mutation is likely of multiple origins in the Chinese population. Among the origins, more new HTT mutations may arise from the (CCG)10 than from other CCG alleles, which suggests that the (CCG)10 allele may represent a predisposing background for CAG expansion in Chinese populations. Therefore, in comparison with Europeans, the significantly lower prevalence of Huntington's disease in Chinese individuals may not be due to the absence of the predisposing background for CAG expansion but instead may partly result from the lower frequency of the predisposing haplotype for CAG instability in the population.

GCH1 mutation and clinical study of Chinese patients with dopa-responsive dystonia.

Dopa-responsive dystonia (DRD) is typically caused by heterozygous mutations in GTP cyclohydrolase 1 gene (GCH1). Our aim was to investigate the clinical and genetic features of Chinese DRD patients. We analyzed a cohort of Chinese DRD patients' clinical data. Mutation of the GCH1 gene was screened by direct sequencing. Additionally, multiplex ligation-dependent probe amplification (MLPA) assay targeting the GCH1 and the TH gene to evaluate large exon deletion or duplicate mutation of the genes were performed in point mutation-negative patients. Ten sporadic DRD patients and two pedigrees including six patients were included in the study. The onset age ranged from 3 to 15 years old. All patients initially presented with walking problems due to lower limb dystonia. The delay between onset and diagnosis ranged from 1 to 42 years old. The symptoms were completely or near-completely abolished with low dose levodopa treatment (dosages ranged from 25 mg to 400 mg/day). Direct sequencing in 14 patients found two known mutations (Gly203Arg in exon 5 in four unrelated patients and Met102Lys in exon 1 in one patient) and one new mutation (Thr186Ile mutation in exon 5 in two unrelated pedigrees). A heterozygous exon 2 deletion in the GCH1 gene was found in one of three point mutation-negative patients by MLPA analysis. Our clinical findings in DRD patients were consistent with other studies. GCH1 gene mutations were quite common in Chinese patients. MPLA should be performed in routine deletion analysis of GCH1 in point mutation-negative DRD patients.

Some single-nucleotide polymorphisms of the TSSK2 gene may be associated with human spermatogenesis impairment.

Tssk2, a member of the testis-specific serine/threonine kinase (TSSK) family, is expressed predominantly in the testis and is crucial for the formation and function of sperm cells in the mouse. Targeted deletion of Tssk1 and Tssk2 in male chimeric mice caused infertility because of haploinsufficiency of the genes. Therefore, it is reasonable to postulate that mutations in the human homolog TSSK2 gene may also play a role in impaired spermatogenesis in humans. To explore the possible association between mutations in the TSSK2 gene and idiopathic infertility in humans, mutation screening of the gene in 494 patients with azoospermia or severe oligozoospermia and 357 fertile controls was performed using denaturing high-performance liquid chromatography and DNA sequencing. As a result, 4 single-nucleotide transitions were identified, including c.80A>G (rs3747052), c.774C>T (rs1052756), c.839C>T (rs1052763), and c.1026G>A (rs1052773). Among them, significant differences in polymorphism frequencies were observed of c.80A>G (rs3747052) and c.774C>T (rs1052756) between the patients and controls; the allele G of c.80A>G (rs3747052) and allele T of c.774C>T (rs1052756) seem to be risk factors for the development of spermatogenic impairment, suggesting that the TSSK2 gene may be associated with male idiopathic infertility in humans.

Phenotypic expression of partial AZFc deletions is independent of the variations in DAZL and BOULE in a Han population.

DAZ on the Y chromosome and 2 autosomal ancestral genes DAZL and BOULE are suggested to represent functional conservation in spermatogenesis. The partial AZFc deletion, a common mutation of the Y chromosome, always involves 2 DAZ copies and represents a different spermatogenic phenotype in the populations studied. To investigate whether the variations in DAZL and BOULE influence partial AZFc deletion phenotype, the genotyping of 15 loci variations, including 4 known mutations and 11 single-nucleotide polymorphisms (SNPs), was carried out in 157 azoo-/oligzoospermic men and 57 normozoospermic men, both groups with partial AZFc deletions. The frequencies of the alleles, genotypes, and haplotypes of the variations were compared between the 2 groups. As a result, for 9 exonic variations in DAZL and BOULE, only T12A was observed in both groups with similar frequency, and I71V was identified in an azoospermic man with b2/b3 deletion, whereas the rest were absent in the population. The distribution of DAZL haplotypes from 4 variations, including T12A, and of BOULE haplotypes from 2 SNPs was similar between men with normozoospermia and spermatogenic failure. Our findings indicate that the contribution of DAZL and BOULE variations to spermatogenic impairment in men with the DAZ defect is greatly limited, suggesting that expression of spermatogenic phenotypes of partial AZFc deletions is independent of the variations in DAZL and BOULE in the Han population.

[Copy number variation of trinucleotide repeat in dynamic mutation sites of autosomal dominant cerebellar ataxias related genes].

OBJECTIVE: To standardize the experimental procedure of the gene test for autosomal dominant cerebellar ataxias (ADCA), and provide the basis for quantitative criteria of the dynamic mutation of spinocerebellar ataxia (SCA) genes in Chinese population. METHODS: Genotyping of the dynamic mutation loci of the SCA1, SCA2, SCA3, SCA6 and SCA7 genes was performed, using florescence PCR-capillary electrophoresis followed by DNA sequencing, to investigate the variation range of copy number of CAG tandem repeat of the genes in 263 probands of ADCA pedigrees and 261 non-related normal controls. Based on the sequencing result, the bias of the CAG copy number estimation using capillary electrophoresis with different DNA controls was compared to analyze the technical detailes of the electrophresis method in testing the dynamic mutation sites. RESULTS: PCR products containing dynamic mutation loci of the SCA genes showed significantly higher mobility than that of molecular weigh marker with relatively balanced GC content. This was particularly obvious in the SCA2, SCA 6 and SCA7 genes whereas the deviation of copy number could be corrected to +/-1 when known CAG copy number fragments were used as controls. The mobility of PCR products was primarily related to the copy number of CAG repeat when the fragments contained normal CAG repeat. In the 263 ADCA pedigrees, 6 (2.28%) carried SCA1 gene mutation, 8 (3.04%) had SCA2 mutation and 81 (30.80%) harbored SCA3 mutation. The gene mutation of SCA6 and SCA7 was not found. The normal variation range of the CAG repeat was 17-36 copies in SCA1 gene, 13-30 copies in SCA2, 14-39 copies in SCA3, 6-16 copies in SCA6 and 6-13 copies in SCA7. The heterozygosity was 76.1%, 17.7%, 74.4%, 72.1% and 41.3%, respectively. The mutation range of the CAG repeat was 49-56 copies in SCA1 gene, 36-41 copies in SCA2, 59-81 copies in SCA3. Neither homozygous mutation of an SCA gene nor double heterozygous mutation of the SCA genes was observed in the study. CONCLUSION: The copy number of the CAG repeat in SCA genes could be calculated accurately based on the result of florescence PCR-capillary electrophoresis when limited amount of known repeat copy number controls were used. Our result supported that the notion that SCA3 gene mutation was the most common cause for ADCA, and the obtained data would be helpful for establishing quantitative criteria of the dynamic mutation of the SCA genes in Chinese.

Y chromosome haplogroups may confer susceptibility to partial AZFc deletions and deletion effect on spermatogenesis impairment.

BACKGROUND: Partial AZFc deletions related to testis-specific gene families are common mutations of the Y chromosome, but their contribution to spermatogenic impairment is still unresolved, and the risk factors for the formation of the deletions remain unknown. With this in mind, we investigated the possible association between Y chromosome haplogroups and predisposition to partial AZFc deletions and their effect on spermatogenesis in a Chinese population. METHODS: The haplogrouping was carried out using 12 polymorphic loci on the Y chromosome in 269 non-AZFc-deleted controls with an unknown spermatogenic status and 214 men with a partial AZFc deletion defined by the absence of the sequence-tagged site and sequence family variant loss of the DAZ and CDY1 genes. In the latter group, 57 men had normozoospermia and 157 men had azoo/oligozoospermia. Among these, 122 had a de novo partial AZFc deletion. RESULTS: Y haplogroup distribution differed significantly between men with a de novo partial AZFc deletion and the control group, and between men with a specific subtype of the partial AZFc deletions and the control group. Further, partial AZFc deletions gave rise to spermatogenesis impairment in some Y haplogroups. CONCLUSIONS: The findings indicate that some monophyletic Y chromosomes may be associated with predisposition to specific subtypes of partial AZFc deletion and adverse effect on spermatogenesis. Although these deletions were not confirmed with gene dosage analysis, the results suggest that Y chromosome background is an important factor that affects partial AZFc deletion formation and its contribution to spermatogenic failure.

[Study on the distribution of Y chromosome haplogroups in Sichuan Han population].

OBJECTIVE: To investigate the distribution characteristics of Y chromosome haplogroups in Sichuan Han population. METHODS: Using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP), denatured high performance liquid chromatography (DHPLC) and DNA sequencing methods, 18 Y-chromosome bialletic markers were detected to type Y chromosome haplogroups in 341 unselected men from Sichuan Han population. RESULTS: A total of 14 haplogroups were observed, in which haplogroups H2 and H4 were identified for the first time in Sichuan population, and haplogroups H14 and N* were found firstly in Chinese. There was a significant frequency difference of Y haplogroups between Sichuan Han population and Southern Han population. CONCLUSION: More comprehensive frequency distribution data of Y chromosome haplogroups are obtained in Sichuan Han population, which would be helpful for understanding the association of Y chromosome background and the susceptibility to male specific diseases such as spermatogenic failure, prostate cancer, testical cancer and so on in present population.