Genotyping of single nucleotide polymorphism in MDM2 genes by universal fluorescence primer PCR and capillary electrophoresis

Single nucleotide polymorphism (SNP) 309 in the promoter region of the murine double minute 2 (MDM2) gene plays an important role in human tumorigenesis. We established a simple and effective CE method for SNP detection in the MDM2 gene. We designed one universal fluorescence-based nonhuman-sequence primer and one fragment-oriented primer, which were combined in one tube, and proceeded with the polymerase chain reaction (PCR). The amplicons were analyzed by capillary electrophoresis using single-strand conformation polymorphism method. PCR fragments generated from this two-in-one PCR displayed either T/T or G/G homozygosity or T/G heterozygosity. A total of 304 samples were blindly genotyped using this developed method, which included the DNA from 138 healthy volunteers, 43 chronic myeloid leukemia (CML) patients, and 123 colorectal cancer (CRC) patients. The results were confirmed by DNA sequencing and showed good agreement. The SSCP-CE method was feasible for SNP screening of MDM2 in large populations.     

变性高效液相色谱法在α-血红蛋白稳定蛋白基因检测中的应用

建立了采用变性高效液相色谱(DHPLC)对α-血红蛋白稳定蛋白(AHSP)基因进行基因分型和突变筛查的新方法。将AHSP基因序列分成6个片段,因第一、二、四、六个片段均含有1～2个常见单核苷酸多态性(single nucleotide polymorphism, SNP)位点,需单个标本分别进行检测;第三、五个片段不含有常见SNP位点,采用DHPLC结合DNA(脱氧核糖核酸)池的方法进行检测。以基因测序为金标准对所建立的AHSP基因检测方法进行方法学评价,结果显示: 40个样品的DHPLC检测结果与测序结果之间完全吻合,说明所建立的检测方法能对AHSP基因6种常见SNP进行准确基因分型。应用DHPLC对365个样品的AHSP基因进行检测,发现2个罕见SNP(11810 G＞A和12802 C＞T);同时还发现2个错义突变(AHSP D29V和AHSP V56G), AHSP D29V突变为新突变,AHSP V56G为罕见突变。结果表明采用DHPLC法可有效地对AHSP基因进行基因分型和突变筛查。     

Analysis of the variations in IL-28RA gene and their association with allergic rhinitis

IL-28RA is one of the important candidate genes for complex trait of genetic diseases, but there is no published information of the genetic variation in this gene. We scanned the seven exons and their boundary introns sequence of IL-28RA including the promoter regions to analyze genetic variation sites, and identified eighteen single nucleotide polymorphisms (SNPs) and two variation sites. We chose seven SNPs (g.-1193 A>C, g.-30 C>T, g.17654 C>T, g.27798 A>G, g.31265 C>T, g.31911 C>T and g.32349 G>A) of them for large sample size genotyping, and assessed the association of genotype and allele frequencies of these SNPs between allergic rhinitis patients and non-allergic rhinitis controls. We also compared the genotype frequencies between Korean controls and Han Chinese control or Korean Chinese control. We investigated the frequencies of haplotype constructed by these SNPs between allergic rhinitis patients and non-allergic rhinitis controls. Our results suggested that the g.32349 G>A polymorphism of IL-28RA might be associated with susceptibility to allergic rhinitis (P=0.032), but seems to have no relationship with serum total IgE levels. The haplotype frequencies by these SNPs also show significant association between controls and allergic rhinitis patients.     

Ribosomal protein mutations in Korean patients with Diamond-Blackfan anemia

Diamond-Blackfan anemia (DBA) is a congenital bone marrow failure syndrome characterized by hypoproliferative anemia, associated physical malformations and a predisposition to cancer. DBA has been associated with mutations and deletions in the large and small ribosomal protein genes, and genetic aberrations have been detected in ∼50–60% of patients. In this study, nine Korean DBA patients were screened for mutations in eight known DBA genes (RPS19, RPS24, RPS17, RPS10, RPS26, RPL35A, RPL5 and RPL11) using the direct sequencing method. Mutations in RPS19, RPS26 and RPS17 were detected in four, two and one patient, respectively. Among the mutations detected in RPS19, two mutations were novel (c.26T>A, c.357-2A>G). For the mutation-negative cases, array-CGH analysis was performed to identify copy-number variations, and no deletions involving the known DBA gene regions were identified. The relative mRNA expression of RPS19 estimated using real-time quantitative PCR analysis revealed two- to fourfold reductions in RPS19 mRNA expression in three patients with RPS19 mutations, and p53 protein expression analysis by immunohistochemistry showed variable but significant nuclear staining in the DBA patients. In conclusion, heterozygous mutations in the known DBA genes RPS19, RPS26 and RPS17 were detected in seven out of nine Korean DBA patients. Among these patients, RPS19 was the most frequently mutated gene. In addition, decreased RPS19 mRNA expression and p53 overexpression were observed in the Korean DBA patients, which supports the hypothesis that haploinsufficiency and p53 hyperactivation represent a central pathway underlying the pathogenesis of DBA.     

Genotyping of single nucleotide polymorphism in γ-glutamyl hydrolase gene by capillary electrophoresis

The γ-glutamyl hydrolase (GGH) gene plays an important role in methotrexate (MTX) metabolism, ensuring that MTX polyglutamates (MTX-(Glu)(n)) could be converted back into MTX. Accumulation of MTX-(Glu)(n) is a problem in MTX therapy. SNP 452 C>T has been reported to associate with lower catalytic activity and higher accumulation of long-chain MTX-(Glu)(n) in patients treated with higher doses of MTX treatment. We propose and establish a simple and effective CE method for detecting SNP in GGH gene. The DNA samples after amplification were analyzed by SSCP-CE method. The CE conditions were generated by using 1× TBE buffer containing 1.5%?w/v hydroxypropyl methyl cellulose under reverse polarity at 25°C. This method was applied to detect genotyping of acute lymphoblastic leukemia patients receiving MTX treatment. The results were confirmed by DNA sequencing with good agreement. Concentrations of MTX-(Glu)(n) in whole blood were analyzed by on-line stacking CE method. MTX-(Glu)(n) levels and genotypes in GGH gene of acute lymphoblastic leukemia patients were evaluated. The SSCP-CE method was found to be feasible for SNP screening in the GGH gene.     

Effects of a chemical chaperone on genetic mutations in ��-galactosidase A in Korean patients with Fabry disease

Fabry disease is an X-linked inborn error of glycosphingolipid catabolism that results from mutations in the gene encoding the α-galactosidase A (GLA) enzyme. We have identified 15 distinct mutations in the GLA gene in 13 unrelated patients with classic Fabry disease and 2 unrelated patients with atypical Fabry disease. Two of the identified mutations were novel (i.e., the D231G missense mutation and the L268delfsX1 deletion mutation). This study evaluated the effects of the chemical chaperones 1-deoxygalactonojirimycin (DGJ) on the function of GLA in vitro, in cells containing missense mutations in the GLA gene. Nine missense and a nonsense mutations, including one novel mutation were cloned into mammalian expression vectors. After transient expression in COS-7 cells, GLA enzyme activity and protein expression were analyzed using fluorescence spectrophotometry and Western blot analysis, respectively. DGJ enhanced GLA enzyme activity in the M42V, I91T, R112C and F113L mutants. Interestingly, the I91T and F113L mutations are associated with the atypical form of Fabry disease. However, DGJ treatment did not have any significant effect on the GLA enzyme activity and protein expression of other mutants, including C142W, D231G, D266N, and S297F. Of note, GLA enzyme activity was not detected in the novel mutant (i.e., D231G), although protein expression was similar to the wild type. In the absence of DGJ, the E66Q mutant had wild-type levels of GLA protein expression and approximately 40% GLA activity, indicating that E66Q is either a mild mutation or a functional single nucleotide polymorphism (SNP). Thus, the results of this study suggest that the chemical chaperone DGJ enhances GLA enzyme activity and protein expression in milder mutations associated with the atypical form of Fabry disease.     

Mutation analysis of PAH gene and characterization of a recurrent deletion mutation in Korean patients with phenylketonuria

Phenylketonuria (PKU; MIM 261600) is an autosomal recessive metabolic disorder caused by a deficiency of phenylalanine hydroxylase (PAH; EC 1.14.16.1). Point mutations in the PAH gene are known to cause PKU in various ethnic groups, and large deletions or duplications account for up to 3% of the PAH mutations in some ethnic groups. However, a previous study could not identify approximately 14% of the mutant alleles by sequence analysis in Korean patients with PKU, which suggests that large deletions or duplication might be frequent causes of PKU in Koreans. To test this hypothesis, we performed multiplex ligation-dependent probe amplification (MLPA) for the identification of uncharacterized mutant alleles after PAH sequence analysis of 33 unrelated Korean patients with PKU. Bi-directional sequencing of the PAH exons and flanking intronic regions revealed 27 different mutations, including four novel mutations (two missense and two deletion mutations), comprising 57/66 (86%) mutant alleles. MLPA identified a large deletion that encompassed exons 5 and 6 in four patients, another large deletion that extended from exon 4 to exon 7 in one patient, and a duplication of exon 4 in one patient. Chromosomal walking characterized the deletion breakpoint of the most common large deletion that involved exons 5 and 6 (c.456_706+138del). The present study shows that the allelic frequency of exon deletion or duplication is 9% (6/66) in Korean PKU patients, which suggests that these mutations may be frequent causes of PKU in Korean subjects.     

A MELAS syndrome family harboring two mutations in mitochondrial genome

Mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes (MELAS) syndrome is a genetically heterogeneous mitochondrial disorder with variable clinical symptoms. Here, from the sequencing of the entire mitochondrial genome, we report a Korean MELAS family harboring two homoplasmic missense mutations, which were reported 9957T>C (Phe251Leu) transition mutation in the cytochrome c oxidase subunit 3 (COX3) gene and a novel 13849A>C (Asn505His) transversion mutation in the NADH dehydrogenase subunit 5 (ND5) gene. Neither of these mutations was found in 205 normal controls. Both mutations were identified from the proband and his mother, but not his father. The patients showed cataract symptom in addition to MELAS phenotype. We believe that the 9957T>C mutation is pathogenic, however, the 13849A>C mutation is of unclear significance. It is likely that the 13849A>C mutation might function as the secondary mutation which increase the expressivity of overlapping phenotypes of MELAS and cataract. This study also demonstrates the importance of full sequencing of mtDNA for the molecular genetic understanding of mitochondrial disorders.     

Association between interleukin 6 promoter variants and chronic hepatitis B progression

Interleukin 6 (IL6) plays an essential role in the regulation of immune response to chronic disease. In this study, the three known single nucleotide polymorphisms (SNPs) in the IL6 promoter region were genotyped in a large chronic hepatitis B cohort to evaluate the effects of IL6 promoter variants. The single base extension method was used for this genotyping. Haplotypes were constructed by the three SNPs in IL6. Allele frequencies were compared for; i) patients with chronic hepatitis (CH) and chronic carriers vs. chronic hepatis patients with clinical evidence of liver cirrhosis (LC) (i.e., portal hypertension), ii) cirrhotic patients with hepatocellular carcinoma (HCC) vs. without HCC by logistic regression, and iii) with respect to the time intervals from the onset of infection to HCC. Results were analyzed by Cox relative hazard analysis on the assumption that all the patients were infected during early infancy. The frequencies of each SNP were 0.002 (IL6-597 G>A), 0.25 (IL6-572 C>G) and 0.002 (IL6-174 G>C), respectively, in the Korean population (n = 1,046). No significant associations were detected between IL6-572 C>G and chronic hepatitis B outcome in this study; i.e., LC occurrence on CH (OR = 0.16-1.27, P = 0.13- 0.71) and HCC occurrence on LC (OR = 1.04-1.23, P = 0.89-0.60) of heterozygotes and homozygotes for G allele in referent comparison to homozygotes for common allele (C/C genotype), and time interval to HCC (RH = 0.67-1.00; P = 0.14-0.99). In conclusion, there appeared to be no significant associations between IL6 promoter variants and disease outcome in chronic hepatitis B.     

The association of eotaxin-2 and eotaxin-3 gene polymorphisms in a Korean population with ulcerative colitis

The eotaxin gene family (eotaxin, eotaxin-2 and eotaxin-3) have been implicated in the recruitment of eosinophils, basophiles and helper T (Th) 2 lymphocytes that is a central aspect of allergic disease. We previously suggested that Eo2+179T>C and Eo2 +275C>T of the eotaxin-2, and Eo3 +2497T>G of the eotaxin-3 were significantly associated with susceptibility to asthma. To determine whether the single nucleotide polymorphisms (SNPs) of eotaxin-2 and eotaxin-3 gene family are associated with the susceptibility of ulcerative colitis (UC), we analyzed the genotype of 119 patients with UC and 303 controls using single-base extension (SBE) method. We also calculated the haplotype frequencies among Eo2 +179T>C and Eo2 +275C >T of the eotaxin-2 and Eo3 +2497T>G of the eotaxin-3 in both control and UC patients. The genotype frequency of Eo2 +179T>C and Eo2 +275C>T between UC patients and controls were significantly different (P=0.006 and 0.022, respectively). The genotype and allele frequencies of EoA2497T>G in UC patients were not significantly different from those in the controls without UC patients. Our results suggest that Eo2 +179T>C and Eo2 +275C>T of eotaxin-2 might be associated with the susceptibility of UC.     

Targeted exome sequencing resolves allelic and the genetic heterogeneity in the genetic diagnosis of nephronophthisis-related ciliopathy

Nephronophthisis-related ciliopathy (NPHP-RC) is a common genetic cause of end-stage renal failure during childhood and adolescence and exhibits an autosomal recessive pattern of inheritance. Genetic diagnosis is quite limited owing to genetic heterogeneity in NPHP-RC. We designed a novel approach involving the step-wise screening of Sanger sequencing and targeted exome sequencing for the genetic diagnosis of 55 patients with NPHP-RC. First, five NPHP-RC genes were analyzed by Sanger sequencing in phenotypically classified patients. Known pathogenic mutations were identified in 12 patients (21.8%); homozygous deletions of NPHP1 in 4 juvenile nephronophthisis patients, IQCB1/NPHP5 mutations in 3 Senior–Løken syndrome patients, a CEP290/NPHP6 mutation in 1 Joubert syndrome patient, and TMEM67/MKS3 mutations in 4 Joubert syndrome patients with liver involvement. In the remaining undiagnosed patients, we applied targeted exome sequencing of 34 ciliopathy-related genes to detect known pathogenic mutations in 7 (16.3%) of 43 patients. Another 18 likely damaging heterozygous variants were identified in 13 NPHP-RC genes in 18 patients. In this study, we report a variety of pathogenic and candidate mutations identified in 55 patients with NPHP-RC in Korea using a step-wise application of two genetic tests. These results support the clinical utility of targeted exome sequencing to resolve the issue of allelic and genetic heterogeneity in NPHP-RC.     

Evaluation of a Apo-1/Fas promoter polymorphism in Korean stroke patients

Apoptosis has been implicated in the pathogenesis of neurodegenerative diseases such as stroke and Alzheimer's disease. Apo-1/Fas gene is one of the mediators of apoptosis in stroke. Mval polymorphism is the first polymorphic marker identified in the Apo-1/Fas gene promoter, which was typed by PCR and followed by Mval digestion and gel electrophoresis. DNA isolated from peripheral blood collected from 91 stroke patients and 103 healthy blood donors was used for genotypes of GG, GA and AA by sequence specific primer PCR. Mval polymorphism was examined based on Fas gene promotor region by restriction fragment length polymorphism (RFLP). The Fas-GG genotype was the least frequent in patients with stroke and healthy controls (P = 0.57). In normal Korean controls the Mval polymorphism GA, AA and GG were 48.6%, 34.9% and 16.5%. In stroke patients were 56.2%, 29.6% and 14.2% respectively. And the allelic frequencies of Mval*2 (G) allele were less frequent than Mval*1 (A) allele in patients with stroke and healthy controls (P = 0.76). In normal Korean controls Mval*1 (A) and Mval*2 (G) alleles were 59.2% and 40.8%. In stroke patients were 57.6% and 42.4%, respectively. Our results, pending confirmation in a larger study, indicate that the Fas genotype may not appear to be a risk factor for stroke in Korean stroke patients.     

Mutational analysis of whole mitochondrial DNA in patients with MELAS and MERRF diseases

Mitochondrial diseases are clinically and genetically heterogeneous disorders, which make the exact diagnosis and classification difficult. The purpose of this study was to identify pathogenic mtDNA mutations in 61 Korean unrelated families (or isolated patients) with MELAS or MERRF. In particular, the mtDNA sequences were completely determined for 49 patients. From the mutational analysis of mtDNA obtained from blood, 5 confirmed pathogenic mutations were identified in 17 families, and 4 unreported pathogenically suspected mutations were identified in 4 families. The m.3243A>G in the tRNA^Leu(UUR) was predominantly observed in 10 MELAS families, and followed by m.8344A>G in the tRNA^Lys of 4 MERRF families. Most pathogenic mutations showed heteroplasmy, and the rates were considerably different within the familial members. Patients with a higher rate of mutations showed a tendency of having more severe clinical phenotypes, but not in all cases. This study will be helpful for the molecular diagnosis of mitochondrial diseases, as well as establishment of mtDNA database in Koreans.     

Genotyping single nucleotide polymorphisms using intact polymerase chain reaction products by electrospray quadrupole mass spectrometry

Both single nucleotide polymorphisms (SNPs) and mutations are commonly observed in the gene encoding the tumor suppressor protein, p53. SNPs occur at specific locations within genes whereas mutations may be distributed across large regions of genes. When determining nucleotide differences, mass spectrometry is the only method other than Sanger sequencing which offers direct structural information. Electrospray ionization (ESI) quadrupole mass spectrometry (MS) analysis of intact polymerase chain reaction (PCR) products was performed following a simple purification and on-line heating to limit ion adduction. The PCR products were amplified directly from genomic DNA rather than plasmids, as in our previous work. Two known polymorphisms of the p53 gene were genotyped. A cytosine (C) or guanine (G) transversion, designated C <--> G (G <--> C on the opposite strand), were each detected by a 40.0 Da change upon ESI quadrupole MS analysis. Using known PCR products as standards, the genotypes determined for 10 human samples corresponded with restriction fragment length polymorphism (RFLP) analysis. Cytosine/thymine (T) transitions, designated C <--> T (G <--> A on the opposite strand), were also genotyped by ESI-MS. This SNP is discriminated by a 15.0 Da change on one strand (C <--> T) and a 16.0 Da change on the other (G <--> A). Appropriate sample preparation and instrumental configuration (including heated sample inlet syringe and MS source), to limit adducts, are both vital for successful ESI quadrupole MS analysis of intact PCR products.     

Single nucleotide polymorphism detection in the hMSH2 gene using conformation-sensitive CE

CE allows for highly reproducible analysis of DNA fragments which can be used to detect DNA mutations including SNPs. We have utilized a simple and direct CE analysis method for SNP analysis called conformation-sensitive CE (CSCE), based on the principle of single nucleotide different to produce conformational changes in the mildly denaturing solvent system. This method was applied to analysis of a mutation in the promoter region of the hMSH2 gene. This gene belongs to the human DNA mismatch repair system, which is responsible for recognizing and repairing mispaired nucleotides, and mutations in the hMSH2 gene are known to cause hereditary nonpolyposis colorectal cancer (HNPCC). PCR fragments generated from the promoter region of the hMSH2 gene, displaying either a C/C homozygote, C/T heterozygote, or T/T homozygote genotype, did not require further pretreatment before electrokinetic injection. The CE separation, using a 1xTris-borate-EDTA (TBE) buffer containing 3% w/v hydroxylethyl cellulose (HEC) and 6 M urea, was performed under reverse polarity with a separation temperature of 15 degrees C. The genotypes of 204 healthy volunteers and 13 colorectal cancer patients were determined using CSCE, and the results confirmed by DNA sequencing. While the CSCE separations were shown to be highly reproducible and sensitive for screening large populations, no correlation was observed between cancer patients and this hMSH2 gene polymorphism.     

A palladium-platinum bimetal nanodendritic melamine network for signal amplification in voltammetric sensing of DNA

A sandwich-type electrochemical DNA sensor is described for the detection of oligonucleotides typical for MECP2 gene mutations. Palladium nanoparticles (PdNPs) and platinum nanoparticles (PtNPs) were used to synthesize flower-like PdPt nanodendrites (NDs) by a one-pot method. The PdPt NDs possess a high specific surface area and excellent catalytic capabilities. They served as the carrier for the signal DNA probe (SP) and simultaneously catalyze the reduction of hydrogen peroxide (H₂O₂). The PdPt NDs were modified with melamine, and this results in the formation of a PdPt-melamine network through stable interactions between the PdPt NDs and the three amino groups of each melamine molecule. The network exhibits excellent catalytic ability in enhancing the current signal response in the voltammetric detection of MECP2 gene mutation, best measured at ?0.4 V vs. SCE and using H₂O₂ as the electrochemical probe. In addition, gold nanoflowers were electrodeposited on the electrode interface in order to accelerate electron transfer and to capture the capture probe. The sensor is stable and can detect MECP2 gene mutations in the 1 fmol·L^?1 to 1 nmol·L^?1 concentration range, with a 0.33 fmol·L^?1 lower detection limit at an S/N ratio of 3.

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Graphical abstract Schematic presentation of electrodes for the determination of the X-linked gene methyl-CpG-binding protein 2 (MECP2). The sensor is based on the electrooxidation of added H₂O₂ by using the melamine modified palladium platinum bimetal nanodendrites as network signal amplification strategy. This versatile platform expands studies on the detection of monogenic disease.

     

Genetic and expression analysis of all non‐synonymous single nucleotide polymorphisms in the human deoxyribonuclease I‐like 1 and 2 genes

Members of the human DNase I family, DNase I‐like 1 and 2 (DNases 1L1 and 1L2), with physiological role(s) other than those of DNase I, possess three and one non‐synonymous SNPs in the genes, respectively. However, only limited population data are available, and the effect of these SNPs on the catalytic activity of the enzyme remains unknown. Genotyping of all the non‐synonymous SNPs was performed in three ethnic groups including six different populations using the PCR‐RFLP method newly developed. Asian and African groups including Japanese, Koreans, Ghanaians and Ovambos were typed as a single genotype at each SNP, but polymorphism at only SNP V122I in DNase 1L1 was found in Caucasian groups including Germans and Turks; thus a Caucasian‐specific allele was identified. The DNase 1L1 and 1L2 genes show relatively low genetic diversity with regard to these non‐synonymous SNPs. The level of activity derived from the V122I, Q170H and D227A substituted DNase 1L1 corresponding to SNPs was similar to that of the wild‐type, whereas replacement of the Asp residue at position 197 in the DNase 1L2 protein with Ala, corresponding to SNP D197A, reduced its activity greatly. Thus, SNP V122I in DNase 1L1 exhibiting polymorphism exerts no effect on the catalytic activity, and furthermore SNP D197A in DNase 1L2, affecting its catalytic activity, shows no polymorphism. These findings permit us to postulate that the non‐synonymous SNPs identified in the DNase 1L1 and 1L2 genes may exert no influence on the activity levels of DNases 1L1 and 1L2 in human populations.     

Identification of RFLP G6PD mutations by using microcapillary electrophoretic chips (Experion)

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is one of the most well-known human genetic defects, identified in more than 400 million individuals in the world. To date, no commercial kits are available for the mutation screening of this disease. Seventy G6PD-deficient Italian individuals admitted to the Laboratory of Clinical Molecular Biology of Hospital "Agostino Gemelli" of Rome were screened for the most frequent Italian mutations, by means of allele-specific PCR, followed by restriction fragment length electrophoresis. The present study compares two techniques for the identification of restriction patterns: agarose gel electrophoresis versus Experion system. When the first screening was negative, the entire G6PD gene was sequenced using the ABI 3100 Avant Instrumentation. The G6PD variants identified and their frequencies were the following: G6PD Mediterranean (75.7%), G6PD Seattle (7.1%), G6PD A(-) 202 + 376 (7.1%), and G6PD Cassano (2.8%). In addition, we identified by direct sequencing two new mutations, namely Buenos Aires and Rignano. With the Experion method, the size band determination was more accurate than that obtained by gel electrophoresis. The Experion system resulted as a valid, easy, and reproducible diagnostic method for the screening of G6PD mutation as compared with the agarose electrophoretic analysis.