OBJECTIVE: To explore the genetic basis for a patient featuring multiple carboxylase deficiency (MCD). METHODS: PCR and Sanger sequencing were used to detect variant in the coding region of BT and HLCS genes in the patient. Suspected variants were verified in her parents and 80 unrelated healthy controls by a PCR-restriction fragment length polymorphism (PCR-RFLP) method. RESULTS: The patient was found to carry compound heterozygous variants of the HLCS gene, namely c.286delG (p.Val96Leufs*162) and c.1648G>A (p.Val550Met). The c.286delG (p.Val96Leufs*162) was verified to be novel variant based on the result of PCR-RFLP analysis. No variant was found in the coding regions of BT gene in the patient. CONCLUSION The compound c.286delG (p.Val96Leufs*162) and c.1648G>A (p.Val550Met) variants probably underlie the MCD disorder in this patient. Above results have enriched the variant spectrum of MCA.
Carbon-Nitrogen Ligases ; genetics ; Exons ; Female ; Humans ; Multiple Carboxylase Deficiency ; genetics ; Mutation ; Open Reading Frames ; Polymerase Chain Reaction ; Polymorphism, Restriction Fragment Length ; Sequence Analysis, DNA

OBJECTIVE: To study the association of the polymorphisms of the serum amyloid A1 (SAA1) gene at rs4638289 and rs7131332 loci with Kawasaki disease (KD) and its complication coronary artery lesion (CAL) in children. METHODS: A total of 105 Han children with KD who were hospitalized and treated from 2013 to 2017 were enrolled as the KD group. A total of 100 Han children who underwent physical examination were enrolled as the control group. According to the presence or absence of CAL, the KD group was further divided into a CAL group with 23 children and a non-CAL (NCAL) group with 82 children. Polymerase chain reaction-restriction fragment length polymorphism was used to investigate the polymorphisms of the SAA1 gene at rs4638289 and rs7131332 loci. RESUKTS: For the locus rs4638289 of the SAA1 gene, there were no significant differences between the KD and control groups in the genotype frequencies of AA, AT, and TT and the allele frequencies of A and T (P>0.05). But there were significant differences between the CAL and NCAL groups in the genotype frequencies of AA, AT, and TT (P=0.016), while there were no significant differences in the allele frequencies of A and T (P>0.05). AT genotype was a protective factor against CAL (OR=0.276, 95%CI: 0.099-0.772, P=0.011). For the locus rs7131332 of the SAA1 gene, there were no significant differences between the KD and control groups in the genotype frequencies of AA, AG, and GG and the allele frequencies of A and G (P>0.05). There were also no significant differences between the CAL and NCAL groups in the genotype frequencies of AA, AG, and GG and the allele frequencies of A and G (P>0.05). CONCLUSIONS Polymorphisms of the SAA1 gene at loci rs4638289 and rs7131332 are not associated with the onset of KD, while the polymorphism at the locus rs4638289 is associated with CAL in KD patients. KD patients with genotype AT may have a reduced risk of CAL.
Case-Control Studies ; Child ; Gene Frequency ; Genetic Predisposition to Disease ; Genotype ; Humans ; Mucocutaneous Lymph Node Syndrome ; genetics ; Polymorphism, Restriction Fragment Length ; Polymorphism, Single Nucleotide ; Serum Amyloid A Protein ; genetics

OBJECTIVE: There is asingle nucleotide polymorphism (SNP) in the exon 2 of the vitamin D receptor (VDR) gene that can be distinguished using the restriction endonuclease FokI, and accordingly divided into three genotypes: FF, Ff and ff. VDR-FokI polymorphism was the only known SNP that could alter the protein structure of VDR. CYP24A1 is the gene encoding vitamin D 24 hydroxylase and is a vitamin D responsive gene. The influence of rs2228570 on transcriptional activation by VDR in human gingival fibroblasts (hGF) and periodontal ligament cells (hPDLC) was investigated in this study. METHODS: hGF and hPDLC of 12 donors' were primarily cultured and genomic DNA was extracted. A part of genomic DNA with the length of 267 bp was obtained using PCR, which contained the SNP. VDR-Fok I genotypes were determined according to the results of restriction fragment length polymorphism. hGF and hPDLC were stimulated with 10 nmol/L 1α,25 dihydroxy vitamin D₃ (1,25OH₂D₃) or 1 000 nmol/L 25 hydroxy vitamin D₃ (25OHD₃) for 48 h before RNA was extracted. Then VDR antagonist ZK159222 was used or not used during 1,25OH₂D₃ or 25OHD₃ stimulation with hGF and hPDLC. After 1,25OH₂D₃ stimulation for 48 h, the proteins in hGF and hPDLC were also collected. The protein expressions of CYP24A1 and VDR were detected using Western blot. RESULTS: Among the 12 donors' cell cultures, the number of FF, ff and Ff genotypes was 4, 3 and 5, respectively.After stimulation with 1,25OH₂D₃ or 25OHD₃ for 48 h,CYP24A1 mRNA levels in FF-hGF were significantly higher than those in other hGF genotypes(1,25OH₂D₃: F=31.147, P<0.01; 25OHD₃: F= 32.061,P <0.01), as was in FF-hPDLC (1,25OH₂D₃: F=23.347, P<0.01; 25OHD₃: F=32.569,P<0.01). When ZK159222 was used before 1,25OH₂D₃ stimulation, this statistically significant difference disappeared (hGF: F=0.246, P=0.787; hPDLC: F=0.574, P=0.583). When ZK159222 was used before 25OHD₃ stimulation, the trend was similar (hGF: F=1.636, P=0.248; hPDLC: F=0.582, P=0.578).After stimulation with 1,25OH₂D₃ for 48 h, CYP24A1 protein levels in FF-hGF were significantly higher than those in the other hGF genotypes (F=12.368, P <0.01), as was in FF-hPDLC (F=15.749, P <0.01). In hGF and hPDLC, the mRNA or protein expression of VDR of different genotypes was not significantly different under different stimulation conditions.The paired comparison showed that there was no statistically significant difference between the expression of CYP24A1 in hGF and that in hPDLC under all the stimulation conditions, as was the expression of VDR. CONCLUSION In hGF and hPDLC, the FF-VDR genotype is associated with the more remarkable up-regulation of CYP24A1than the other genotypes, indicating that transcriptional activation of FF-VDR might be higher than those of other vitamin D receptors.
Fibroblasts/metabolism* ; Genotype ; Humans ; Periodontal Ligament/metabolism* ; Polymorphism, Genetic ; Polymorphism, Restriction Fragment Length ; Polymorphism, Single Nucleotide ; Receptors, Calcitriol/genetics* ; Vitamin D3 24-Hydroxylase/metabolism*

OBJECTIVES: To detect the genotype of ABO blood group by SNaPshot technology. METHODS: DNA were extracted from the peripheral blood samples with known blood groups （obtained by serology） of 107 unrelated individuals in Yunnan. Six SNP loci of the 261th, 297th, 681th, 703th, 802th, and 803th nucleotide positions were detected by SNaPshot Multiplex kit, and relevant genetics parameters were calculated. RESULTS: In 107 blood samples, the allele frequencies of types A, B, O^A, and O^G were 0.355 1, 0.168 2, 0.230 0 and 0.247 6, respectively, while that of types A^G and cis AB were not detected. The genotyping results of ABO blood group were consistent with that of serologic testing. CONCLUSIONS SNaPshot technology can be adapted for genotyping of ABO blood group.
ABO Blood-Group System/genetics* ; Alleles ; Asian People/genetics* ; China ; DNA ; Ethnicity ; Gene Frequency ; Genotype ; Humans ; Polymerase Chain Reaction/methods* ; Polymorphism, Restriction Fragment Length

BACKGROUNDG-protein β-polypeptide 3 (GNB3) is a β subunit isoform of G-protein that plays important role in signal transduction of membrane G-protein coupled receptors (GPCRs). The GNB3 splice variant C825T (rs5443) is associated with risk for essential hypertension (EH) and efficacy of therapeutic drugs targeting GPCRs. It is unknown whether the polymorphism is associated with blood pressure (BP) response to telmisartan or amlodipine, two widely prescribed antihypertensive drugs.

METHODSA total of 93 subjects initially diagnosed as EH were recruited and underwent a 4-week treatment with telmisartan (42 patients) or amlodipine (51 patients) monotherapy. Both baseline and after-treatment BP were measured. GNB3 C825T polymorphism was genotyped by polymerase chain reaction-restriction fragment length polymorphism.

RESULTSBaseline systolic BP (SBP) and diastolic BP (DBP) were comparable among C825T genotypes in both telmisartan and amlodipine treatment groups. Patients with the CT or TT genotypes showed significantly lower body mass index (BMI) as compared with CC homozygotes in both groups (P < 0.05, respectively). GNB3 825TT homozygotes showed significantly higher after-treatment DBP and mean arterial pressure (MAP) than those carrying at least one 825C allele (P < 0.01) in the telmisartan treatment group. No difference in after-treatment SBP, DBP, and MAP levels among C825T genotypes was observed in the amlodipine treatment group. No significant difference in absolute changes in BP levels was observed among the genotypes in either treatment group.

CONCLUSIONThe GNB3 C825T splice variant is associated with the DBP-lowering effect of telmisartan but not amlodipine in Chinese EH patients.

Adult ; Aged ; Amlodipine ; therapeutic use ; Antihypertensive Agents ; therapeutic use ; Benzimidazoles ; therapeutic use ; Benzoates ; therapeutic use ; Blood Pressure ; drug effects ; Essential Hypertension ; Female ; Genotype ; Heterotrimeric GTP-Binding Proteins ; genetics ; Humans ; Hypertension ; drug therapy ; genetics ; Male ; Middle Aged ; Polymorphism, Genetic ; genetics ; Polymorphism, Restriction Fragment Length ; genetics

The aim of the present study is to determine the characteristics of genotype and phenotype of Echinococcus granulosus derived from wild sheep and to compare them with the strains of E. granulosus sensu stricto (sheep-dog) and E. granulosus camel strain (camel-dog) in Iran. In Khojir National Park, near Tehran, Iran, a fertile hydatid cyst was recently found in the liver of a dead wild sheep (Ovis orientalis). The number of protoscolices (n=6,000) proved enough for an experimental infection in a dog. The characteristics of large and small hooks of metacestode were statistically determined as the sensu stricto strain but not the camel strain (P=0.5). To determine E. granulosus genotype, 20 adult worms of this type were collected from the infected dog. The second internal transcribed spacer (ITS2) of the nuclear ribosomal DNA (rDNA) and cytochrome c oxidase 1 subunit (COX1) of the mitochondrial DNA were amplified from individual adult worm by PCR. Subsequently, the PCR product was sequenced by Sanger method. The lengths of ITS2 and COX1 sequences were 378 and 857 bp, respectively, for all the sequenced samples. The amplified DNA sequences from both ribosomal and mitochondrial genes were highly similar (99% and 98%, respectively) to that of the ovine strain in the GenBank database. The results of the present study indicate that the morpho-molecular features and characteristics of E. granulosus in the Iranian wild sheep are the same as those of the sheep-dog E. granulosus sensu stricto strain.
Animals ; DNA, Helminth/genetics ; DNA, Ribosomal Spacer/genetics ; Dogs ; Echinococcosis/parasitology/*veterinary ; Echinococcus granulosus/anatomy & histology/classification/genetics/*physiology ; Electron Transport Complex IV/genetics ; *Genotype ; Iran ; *Phenotype ; Phylogeny ; Polymorphism, Restriction Fragment Length ; Sheep ; Sheep Diseases/*parasitology ; Species Specificity

Toxoplasmosis is an important zoonotic disease that can cause abortion in humans and animals. The aim of this study was isolation and subsequent genotyping of Toxoplasma gondii isolates in ovine aborted fetuses. During 2012-2013, 39 ovine aborted fetuses were collected from sheep flocks in Khorasan Razavi Province, Iran. The brain samples were screened for detection of the parasite DNA by nested PCR. The positive brain samples were bioassayed in Webster Swiss mice. The serum samples of mice were examined for T. gondii antibodies by IFAT at 6 weeks post inoculation, and T. gondii cysts were searched in brain tissue samples of seropositive mice. The positive samples were genotyped by using a PCR-RLFP method. Subsequently, GRA6 sequences of isolates were analyzed using a phylogenetic method. The results revealed that T. gondii DNA was detected in 54% (20/37, 95% CI 38.4-69.0%) brain samples of ovine aborted fetuses. In bioassay of mice, only 2 samples were virulent and the mice were killed at 30 days post inoculation, while the others were non-virulent to mice. The size of cysts ranged 7-22 µm. Complete genotyping data for GRA6 locus were observed in 5 of the 20 samples. PCR-RLFP results and phylogenetic analysis revealed that all of the isolated samples were closely related to type I. For the first time, we could genotype and report T. gondii isolates from ovine aborted fetuses in Khorasan Razavi Province, Iran. The results indicate that the T. gondii isolates are genetically related to type I, although most of them were non-virulent for mice.
Aborted Fetus/*parasitology ; Animals ; Brain/parasitology ; Genotype ; Iran ; Mice ; Phylogeny ; Polymerase Chain Reaction ; Polymorphism, Restriction Fragment Length ; Sheep ; Sheep Diseases/*parasitology ; Toxoplasma/classification/*genetics/*isolation & purification/pathogenicity ; Toxoplasmosis, Animal/*parasitology

Laboratory workers, in resource-poor countries, still consider PCR detection of Giardia lamblia more costly and more time-consuming than the classical parasitological techniques. Based on 2 published primers, an in-house one-round touchdown PCR-RFLP assay was developed. The assay was validated with an internal amplification control included in reactions. Performance of the assay was assessed with DNA samples of various purities, 91 control fecal samples with various parasite load, and 472 samples of unknown results. Two cysts per reaction were enough for PCR detection by the assay with exhibited specificity (Sp) and sensitivity (Se) of 100% and 93%, respectively. Taking a published small subunit rRNA reference PCR test results (6%; 29/472) as a nominated gold standard, G. lamblia was identified in 5.9% (28/472), 5.2%, (25/472), and 3.6% (17/472) by PCR assay, RIDA® Quick Giardia antigen detection test (R-Biopharm, Darmstadt, Germany), and iodine-stained smear microscopy, respectively. The percent agreements (kappa values) of 99.7% (0.745), 98.9% (0.900), and 97.7% (0.981) were exhibited between the assay results and that of the reference PCR, immunoassay, and microscopy, respectively. Restriction digestion of the 28 Giardia-positive samples revealed genotype A pattern in 12 and genotype B profile in 16 samples. The PCR assay with the described format and exhibited performance has a great potential to be adopted in basic clinical laboratories as a detection tool for G. lamblia especially in asymptomatic infections. This potential is increased more in particular situations where identification of the parasite genotype represents a major requirement as in epidemiological studies and infection outbreaks.
DNA, Protozoan/genetics ; Developing Countries ; Feces/parasitology ; Genotype ; Giardia lamblia/genetics ; Giardiasis/*diagnosis ; Humans ; Microscopy ; Parasitology/economics/instrumentation/*methods ; *Polymerase Chain Reaction ; *Polymorphism, Restriction Fragment Length ; Reproducibility of Results ; Sensitivity and Specificity

OBJECTIVETo investigate the effects of -260C>T and -651C>T genetic variants in the promoter region of CD14 on the susceptibility to laryngeal cancer.

METHODSA total of 163 patients with laryngeal cancer and 326 healthy subjects as controls were included. Genotypes of CD14 -260C>T (rs2569190) and -651C>T (rs5744455) variants were determined by polymerase chain reaction-based restriction fragment length polymorphism (PCR-RFLP). Odd ratios (OR) and 95% confidence intervals (95%CI) were calculated with logistic regression analysis.

RESULTSCompared with CD14 -651CC genotype carriers, -651TT genotype carriers had the increased risk of laryngeal cancer with the OR (95%CI) of 5.79 (2.38-14.11). When the -651TT genotype carriers were stratified by smoking status, the OR (95%CI) for laryngeal cancer was 8.64 (1.88-39.77) in nonsmokers and 4.74 (1.69-13.25) in smokers; the OR (95%CI) was 5.40 (1.10-26.45) in the light smokers and 4.30 (1.10-16.75) in the hevey smokers. When the -651TT genotype carriers were stratified by drinking status, the OR (95%CI) for laryngeal cancer was 6.48 (2.81-14.95) in nondrinkers and 2.01 (0.65-6.26) in drinkers. There was no significant difference in CD14 -260C>T genotype distribution between patients and controls.

CONCLUSIONPolymorphisms of -651C>T in CD14 promoter contribute to the susceptibility to laryngeal cancer in Chinese population.

Asian Continental Ancestry Group ; Case-Control Studies ; Genetic Predisposition to Disease ; Genotype ; Humans ; Laryngeal Neoplasms ; genetics ; Lipopolysaccharide Receptors ; genetics ; Odds Ratio ; Polymerase Chain Reaction ; Polymorphism, Restriction Fragment Length ; Polymorphism, Single Nucleotide ; Promoter Regions, Genetic

Autism spectrum disorder (ASD) is a neurodevelopmental disorder characterized by high heritability. Recently, autism, the most profound form of ASD, has been increasingly attributed to synaptic abnormalities. Postsynaptic density 95 (PSD95), encoding PSD protein-95, was found essential for synaptic formation, maturation and plasticity at a PSD of excitatory synapse. It is possibly a crucial candidate gene for the pathogenesis of ASD. To identify the relationship between the rs13331 of PSD95 gene and ASD, we performed a case-control study in 212 patients and 636 controls in a Chinese population by using a polymerase chain reaction-restriction fragment length polymerase (PCR-RFLP) assay. The results showed that in genetic analysis of the heterozygous model, an association between the T allele of the rs13331 and ASD was found in the dominant model (OR=1.709, 95% CI 1.227-2.382, P=0.002) and the additive model (OR=1.409, 95% CI=1.104-1.800, P=0.006). Our data indicate that the genetic mutation C>T at the rs13331 in the PSD95 gene is strikingly associated with an increased risk of ASD.
Aged ; Autism Spectrum Disorder ; genetics ; Case-Control Studies ; Child ; Child, Preschool ; China ; Disks Large Homolog 4 Protein ; Female ; Humans ; Intracellular Signaling Peptides and Proteins ; genetics ; Male ; Membrane Proteins ; genetics ; Middle Aged ; Polymorphism, Restriction Fragment Length ; Polymorphism, Single Nucleotide