Mutagenically Separated Polymerase Chain Reaction Research Articles

Gene polymorphisms of Interleukin 6 (\u2212174 G/C) and transforming growth factor \u03b2-1(+915 G/C) in ovarian cancer patients

BackgroundIn the study on hand, we investigated the effect of IL-6 (−174 G/C; rs 1800795) and TGF-β1 (+915G/C; rs 1800471) gene polymorphisms on the susceptibility to Ovarian Cancer and their effect on plasma levels. IL-6 (−174 G/C) SNP was analyzed using mutagenically separated polymerase chain reaction (MS-PCR) while TGF-β1 +915G/C (codon 25) SNP was investigated by the sequence-specific primer polymerase chain reaction (SSP-PCR). An enzyme-linked immunosorbent assay (ELISA) was used to quantify IL-6 and TGF-β1 plasma levels in 48 ovarian cancer patients and 48 normal controls.ResultsRegarding IL 6 (−174 G/C), a significant increase in CC and GC+CC genotypes parallel with the C allele was considered as risk factors for ovarian cancer; on the other hand, the G allele was considered as a protective factor for ovarian cancer. TGF-β1 (+915G/C) investigations showed a significant elevation in GC and GC+CC genotypes which can be considered as a risk factor for ovarian cancer. Plasma IL-6 and TGF-β1 were higher in ovarian cancer patients compared with controls. No specific genotype or allele could be responsible for the elevation of TGF-β1 in ovarian cancer patients’ plasma, while the highest significant value for IL6 in subjects carrying GG and CC genotypes in comparison with GC genotype.ConclusionsThis study supports an association of IL6 (−174G/C) and TGF-β1 (+915G/C) gene polymorphisms with the susceptibility to ovarian cancer.

Cytokine gene polymorphisms in lymphoma: Thesis Abstract

Thesis Abstract Several cytokines are known to be involved in the pathogenesis of non-Hodgkin Lymphomas (NHL). Given the importance of understanding the genetic predisposition involved in the pathogenesis of NHL, this work was designed to study the impact of IL-10 (-1082 G/A; rs1800896 and -819 C/T; rs1800871), IL-6 (-174 G/C; rs1800795) and CD38 (184C/G; rs6449182) gene polymorphisms on the susceptibility of Egyptians to NHL. Both loci of IL10 were genotyped using polymerase chain reaction- sequence-specific primers (PCR-SSP), IL6 rs1800795 G/C was genotyped using mutagenically separated PCR (MS-PCR) and restriction fragment length polymorphism-PCR (RFLP-PCR) was used to genotype CD38 (rs6449182 C/G) in 100 Egyptian patients and 119 controls. Plasma levels of IL-6 and IL-10 were measured using enzyme-linked immunosorbent assay (ELISA). An insignificant change in IL-10 (rs1800896 and rs1800871) genotypes were recorded. GT haplotype was significantly elevated.

Association between IL-18 and IL-6 gene polymorphisms and the risk of T1D in Egyptian children.

To test the involvement between IL-18 and IL-6 genetic polymorphisms and susceptibility to Type 1 diabetes (T1D). Single nucleotide polymorphisms (SNPs) at positions -607A/C and - 137G/C in IL-18 promoter region were examined by sequence specific primers-polymerase chain reaction (SSP-PCR) and position -174G/C in promoter region of IL-6 gene which analyzed by Mutagenically Separated PCR (MS-PCR) in 104 T1D participants and 114 controls. IL-18 -137GC and -137CC genotypes and -137C allele were significantly decreased in T1D subjects (P < 0.05), while -137GG genotype was insignificantly increased as compared to controls. A significant decrease was detected in haplotype -137C/-607C frequency in T1D participants compared with controls (OR = 0.04, P < 0.001). There was significant association between IL-18 -607 of (CC, AC and AA genotypes) in age at diagnosis, glycated hemoglobin (HbA1c) and higher body mass index (BMI) (P < 0.05). This study demonstrated that IL-18 gene promoter polymorphisms might be associated with susceptibility to T1D in Egyptian children. Individuals carrying CC genotype at position -137 of IL-18 promoter may be at a low risk of T1D progression. Additionally, the susceptible combination of IL-18 and IL-6 cytokine genes associated with T1D highlight their risk toward the disease. The online version contains supplementary material available at 10.1007/s40200-021-00763-w.

A novel simple genotyping assay for detection of the ‘Gait keeper’ mutation in DMRT3 and allele frequencies in Azteca and Costa Rican Saddle Horse breeds

The ‘Gait keeper’ mutation in the DMRT3 gene alters locomotion and gait patterns in horses. This mutation (C>A) has been found in all gaited breeds of horses analyzed but is absent in most non-gaited breeds. We developed a new mutagenically separated polymerase chain reaction (MS-PCR) based method for simple detection of horse DMRT3 genotype. Our method was applied in a preliminary study to determine DMRT3 allele frequencies in 78 Azteca horses (AZ) and 53 Costa Rican Saddle Horses (CRSH). We found a wild-type C allele frequency of 100% in the AZ horses. For the CRSH, the wild-type C frequency and mutant A allele frequency were 88.7% and 11.3%, respectively.

IRAK2 is associated with systemic lupus erythematosus risk.

Interleukin-1 receptor-associated kinases (IRAKs) are serine-threonine kinases involved in toll-like receptor and interleukin-1 signaling pathways. They play a key role in inflammation and innate immunity. IRAKs have been previously incriminated in autoimmune diseases such as systemic lupus erythematosus (SLE) and rheumatoid arthritis and inhibition of IRAKs has been recently regarded as a potential therapeutic strategy for SLE. The aim of the present study was to test the association between IRAK2 rs708035 and rs3844283 with SLE. IRAK2 rs708035 and rs3844283 were genotyped by mutagenically separated polymerase chain reaction (MS-PCR) in 142 SLE patients and 149 age- and gender-matched controls. The hyperfunctional IRAK2 rs708035 A allele was more frequent among SLE patients than controls (62.9% versus 54.7%, p = 0.046). IRAK2 rs3844283 C allele was present in 66.5% of patients and 75.5% of controls. The CC genotype was the most frequently exhibited genotype. It was carried by 45.1% of patients with SLE and 57.7% of controls. The G allele was associated with an increased risk of SLE (OR = 1.54, 95%, CI = 1.07-2.22, p = 0.017). IRAK2 rs708035 and IRAK2 rs3844283 were in linkage disequilibrium (D' = 0.64). The AG haplotype was more frequently observed in SLE patients than in controls (0.292 versus 0.194, p = 0.008). This study for the first time ever reveals the association of IRAK2 rs708035 and IRAK2 rs3844283 and the corresponding haplotypes with SLE. Our findings give additional rationale to target IRAKs in the treatment of SLE.Key Points• IRAK2 rs708035 A allele is more frequent in SLE patients than in controls and IRAK2 rs3844283 G allele is associated with SLE susceptibility.• These two alleles are in linkage disequilibrium.• The AG haplotype is associated with SLE.

Interleukin-6 promotor gene polymorphisms and susceptibility to chronic hepatitis B virus in Egyptians

AimTo investigate the association between IL-6 polymorphisms (−174G/C, −572G/C and −597G/A) and susceptibility to chronic hepatitis B virus (CHB) infection. MethodTotal 108 subjects with CHB infection and 102 healthy controls were enrolled in this study. IL-6 (−174G/C) was genotyped using Mutagenically separated Polymerase Chain Reaction (MS-PCR) while sequence specific primers-PCR (SSP-PCR) was used for studying −572G/C and −597G/A. IL-6 plasma level was measured using Enzyme-linked immunosorbent assay (ELISA). ResultsA significant increase (P < 0.01, P < 0.01, P < 0.001) in −174GG, −572GC and −597GA; respectively in the CHB group compared to control group, while −572GG genotype was significantly decreased (P < 0.01) in CHB patients. A significant increase (p < 0.01, p < 0.01) in −174 G and −597A alleles was observed in the CHB patient group; respectively. GGA haplotype is significantly increased (P < 0.05) while GCA haplotype is significantly decreased (P < 0.001) in the patient group. A moderate linkage disequilibrium (LD) (D′ = 0.719, r2 = 0.474; P < 0.001) between IL-6 (−572G/C and −597G/A) was observed. A significant reduction (P < 0.01) in IL-6 plasma level in CHB patients compared to healthy controls (22.28 ± 1.93 versus 32.08 ± 2.41), which was negatively correlated (r = −0.216; P < 0.01) with HBV infection. ConclusionsThis study pointed to the potential role of IL-6 (−174G/C, −572 G/C and −597G/A) gene polymorphisms in the susceptibility to HBV infection. Our results allow for only preliminary conclusions due to relatively small sample size. There is a need for further larger scale studies to fully examine the possible relationship between these cytokine gene polymorphisms and the development of CHB.

Obesity Risk Prediction among Women of Upper Egypt: The impact of FTO rs17817449 gene polymorphism, serum ghrelin and high sensitivity C- reactive protein

Obesity is one of the main threats to the human health. It is a major risk factor for hyperinsulinemia, hypertension, hyperlipidemia, type II diabetes mellitus, and atherosclerotic cardiovascular disease. FTO gene variants have been associated with obesity and diabetes mellitus in different populations, but its role in the susceptibility of these diseases remains unknown. The present study is undertaken to assess the contribution of the FTO rs17817449 gene variants towards obesity and diabetes development and to evaluate the role of ghrelin and hs- CRP on the outcome of obesity in the Upper Egyptian women. A total of 229 subjects, 115 obese (65 non diabetics, 50 diabetics) and 114 non obese non diabetic controls were included in this case control study. Genotyping of FTO gene rs17817449 (T>G) polymorphism was performed by mutagenically separated PCR (MS-PCR) method. Estimation of serum gherlin, hs-CRP levels, related anthropometric and metabolic parameters were performed. The results revealed higher frequency of FTO rs17817449 G allele among obese subjects (46.5%) and obese diabetics (45%) compared to the controls (33.3%) which comprise about 1.75 times increase in the risk for obesity (p<0.01). The distribution of the GG and TG genotypes of FTO were 25.2%, and 42.6% among obese non diabetic, 24% and 42% among obese diabetic and 14.9% and 36.8% among controls respectively. FTO-GG genotype variant was significantly associated with weight, BMI and waist and hip circumference (p<0.05 for each). FTO GG carriers had 2.54 times the possibility to have obesity more than TT carriers. Ghrelin levels were significantly decreased and hs-CRP levels were significantly increased in obese subjects compared to the controls (P<0.001 for each). There was a significant negative correlation between serum ghrelin and hs-CRP (p<0.05). No significant association was detected between FTO genotypes and each of ghrelin, hs-CRP, lipid profile, fasting glucose or insulin levels. In conclusion, the G allele of FTO rs17817449 genotyping is associated with increased obesity risk but there is a lack of association with diabetes. It is also associated with some obesity indices as BMI, hip and waist circumference in the Upper Egyptian women. Both Ghrelin and hs- CRP could play a role in developing obesity. To the best of our knowledge, this is the first study of FTO SNP in Upper Egyptian women. Switching off this FTO faulty gene variant by the recent therapies (as certain foods or gene therapy) will prevent the percentage of women who is affected by this risk allele to get obese via burning rather than storing energy.Key words: FTO gene polymorphism, ghrelin, hs-CRP, Obesity

Micro RNA-146a But Not IRAK1 is Associated with Rheumatoid Arthritis in the Tunisian Population.

Rheumatoid arthritis (RA) is characterized by the production of an array of proinflammatory cytokines through the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signaling pathway. Interleukin-1 receptor (IL-1R) and Toll-like receptors contain a common cytoplasmic motif the Toll/IL-1R (TIR) homology domain. This motif is required for NF-κB activation. IL-1R-associated kinase 1 (IRAK1) is a key adapter molecule recruited during the signaling cascade of the TIR. Its gene expression is regulated by the micro-RNA (miR)-146a. We investigated the role of IRAK1 single-nucleotide polymorphism (SNP) rs3027898 (IRAK1 rs3027898) and miR-146a SNP rs2910164 (miR-146a rs2910164) in Tunisian patients with RA and their association with C reactive protein (CRP), rheumatoid factor (RF), anticyclic citrullinated peptide (anti-CCP) antibodies, and erosion. In a cohort of 172 adult RA patients and 224 matched controls, IRAK1 rs3027898 genotyping was determined by mutagenically separated polymerase chain reaction (MS-PCR) with newly designed primers, and miR-146a rs2910164 genotyping was determined by fragment length polymorphism PCR-restriction (RFLP-PCR). The IRAK1 rs3027898 A allele was detected in 67% of RA patients and 70% of controls indicating that it is not associated with RA in codominant, dominant, or recessive models even after stratification by age and gender. The miR-146a rs2910164 G allele was detected in 76% of RA patients and 68% of controls, thus the C allele confers some protection based on a dominant model [CC+GC (odds ratio (95% confidence interval) = 0.6 (0.3-0.9), p = 0.03)]. No association with CRP, RF, anti-CCP, or erosion was found for either SNPs. The IRAK1 rs3027898 was not associated with RA, whereas C allele of miR-146a rs2910164 was found to be protective. Functional studies are required to investigate the exact role of miR-146a rs2910164 during RA.

New test for endothelin receptor type B (EDNRB) mutation genotyping in horses

Lethal white foal syndrome (LWFS) is an autosomal recessive disease of neonatal foals characterized by a white hair coat and a functional intestinal obstruction. Traditional techniques for identifying the dinucleotide mutation (TC→AG) of the endothelin receptor B gene (EDNRB) associated with LWFS are time-consuming. We developed a new technique based on mutagenically separated polymerase chain reaction (MS-PCR) for simple detection of the EDNRB genotype in horses.

Genetic polymorphisms of interleukin 6 and interleukin 10 in Egyptian patients with systemic lupus eythematosus.

Systemic lupus erythematosus (SLE) is a complex autoimmune disease. Cytokine gene polymorphisms play an important role in SLE. Thus, this study aimed to investigate the associations between interleukin 6 (IL-6) and interleukin 10 (IL-10) promoter single-nucleotide polymorphisms (SNPs) and their susceptibility to SLE and the implications for plasma levels. We genotyped IL-6-174G/C (rs1800795) using mutagenically separated polymerase chain reaction (MS-PCR) and IL-10-1082G/A (rs1800896) and -819C/T (rs1800871) using sequence specific primer polymerase chain reaction (SSP-PCR) in 100 Egyptian patients and 119 controls. The plasma levels of IL-6 and IL-10 were measured by enzyme-linked immunosorbent assay (ELISA). There was significant increase in the frequency of IL-6 (-174) GG genotype (P < 0.05) and G allele (P < 0.01) compared to controls. A significant increase in the distribution of IL-10 (-1082G/A) GG (P < 0.05) and AA (P < 0.05) genotypes and a significant reduction in the frequency of GA genotype (P < 0.05) was found in SLE patients. The mean serum concentration of IL-6 (P < 0.001) and IL-10 (P < 0.001) was significantly elevated in SLE patients compared to healthy controls. There was no significant association of the most common clinical findings and IL-6 and IL-10 gene polymorphisms in SLE patients. In conclusion, our preliminary study indicated that both GG genotype and G allele of IL-6 (-174G/C) could be considered as risk factors for SLE. In addition, the polymorphisms at IL-10 (-1082 G/G and AA) may play a role in SLE susceptibility in Egyptian patients. Larger prospective studies are needed to confirm our findings.

The Methylation Status of MGMT and SFRP2 Genes in Iranian Patients With Polyps

Background: Colorectal cancer is the third most common cancer in the world and the forth cause of death by cancer globally. Colorectal cancer starts with a polyp and might develop due to multiple epigenetic and genetic changes that lead to change of normal colon epithelium to adenocarcinoma of colon. Recent studies show that epigenetic changes such as methylation of SFRP2 (secreted frizzled-related2) a gene in negative regulation Wnt signaling pathways and MGMT (methylguanine-DNA methyltransferase) a DNA repair gene is associated with colorectal cancer. Objectives: Due to correlation of polyp and cancer, the current study aimed to evaluate the role of SFRP2 and MGMT methylation in patients with polyp in south of Iran. Materials and Methods: Forty eight tissue samples of polyp and adenoma, and 20 control samples were investigated for methylation status of SFRP2 and MGMT with mutagenically separated Polymerase Chain Reaction (MS-PCR) method. Results: Methylation in at least one gene was reported about 93.8%. The methylation of SFRP2 was reported in 39 out of the 48 subjects (81.2%) whereas methylation of MGMT was observed in 32 subjects (66.6%). Conclusions: The current study results showed that methylation of SFRP2 and MGMT play an important role in polyp formation.

CD38 and Interleukin 6 Gene Polymorphism in Egyptians with Diffuse Large B-Cell Lymphoma (DLBCL)

Given the importance of understanding the genetic variations involved in the pathogenesis of non-Hodgkin’s lymphoma (NHL), this pilot study was designed to investigate the impact of CD38 (184C/G; rs6449182) and IL-6 (−174 G/C; rs1800795) gene polymorphism on susceptibility of Egyptians to diffuse large B cell lymphoma (DLBCL); major types of NHL. To the best of our knowledge, this study is the first one that examines CD38 polymorphism in the NHL. Genotyping polymorphism is performed using restriction fragment length polymorphism-polymerase chain reaction (RFLP-PCR) for CD38 and Mutagenically separated PCR (MS-PCR) for IL-6 in 100 Egyptian NHL patients with DLBCL subtype and 119 normal controls. The serum level of IL-6 was measured using Enzyme-linked immunosorbent assay (ELISA). CD38 (184C/G) genotype is significantly increased in NHL patients (p < 0.01), while the GG genotype is significantly increased in controls (p < 0.05). Only two genotypes were found (GG and GC) in IL-6 (−174), no CC in our NHL patients and only one case in the controls. Insignificant change in IL-6 (−174 G/C) genotypes was recorded. Significantly increased serum IL-6 (p < 0.05) was positively correlated (r = 0.17; p < 0.05) with the disease. Taken together, our data stressed the importance of CD38 gene polymorphism in developing DLBCL. Our pilot study indicates that CD38 (184) CG genotype might play a role in DLBCL susceptibility in Egyptians. Additional prospective studies on larger population are needed to confirm our findings.

Analysis of Inducible Nitric Oxide Synthase Gene Polymorphisms in Vitiligo in Han Chinese People

BackgroundVitiligo is a chronic depigmented skin disorder with regional melanocytes depletion. The pathogenesis was not completely clarified. Recently, more and more evidence suggested that polymorphisms of some genes are associated with vitiligo risk. Here, we want to examine the association between the inducible nitric oxide synthase (iNOS) gene polymorphisms and the risk of vitiligo in Chinese populations.Methods and Principal FindingsIn a hospital-based case-control study of 749 patients with vitiligo and 763 age- and sex-matched healthy controls, three polymorphisms of iNOS gene were genotyped by using the PCR-restriction fragment length polymorphism (PCR-RFLP) and mutagenically separated PCR (MS-PCR) methods, respectively. We found the iNOS-954 polymorphism was associated with a significantly higher risk of vitiligo (adjusted OR = 1.36, 95% CI = 1.02–1.81). Furthermore, this association is more pronounced in vulgaris vitiligo, active vitiligo and vitiligo without other autoimmune diseases in the stratification study. Analysis of haplotypes showed increased risk for the C -1173 C -954 CEx 16+14 (OR = 1.44, 95% CI = 1.01–1.74). In addition, the serum iNOS activity is significantly associated with iNOS-954 combined genotype (GC+CC) and is much higher in vitiligo patients than in the controls (P<0.01). Logistic regression analysis of iNOS activity showed increased risk between higher activity and iNOS-954 G→C variant genotype carriers (Ptrend<0.001).Conclusions and Significance INOS gene polymorphisms may play an important role in the genetic susceptibility to the development of vitiligo.

Rapid genotyping assays for the 4–base pair deletion of canine MDR1/ABCB1 gene and low frequency of the mutant allele in Border Collie dogs

P-glycoprotein, encoded by the MDR1 or ABCB1 gene, is an integral component of the blood-brain barrier as an efflux pump for xenobiotics crucial in limiting drug uptake into the central nervous system. Dogs homozygous for a 4-base pair deletion of the canine MDR1 gene show altered expression or function of P-glycoprotein, resulting in neurotoxicosis after administration of the substrate drugs. In the present study, the usefulness of microchip electrophoresis for genotyping assays detecting this deletion mutation was evaluated. Mutagenically separated polymerase chain reaction (MS-PCR) and real-time PCR assays were newly developed and evaluated. Furthermore, a genotyping survey was carried out in a population of Border Collies dogs in Japan to determine the allele frequency in this breed. Microchip electrophoresis showed advantages in detection sensitivity and time saving over other modes of electrophoresis. The MS-PCR assay clearly discriminated all genotypes. Real-time PCR assay was most suitable for a large-scale survey due to its high throughput and rapidity. The genotyping survey demonstrated that the carrier and mutant allele frequencies were 0.49% and 0.25%, respectively, suggesting that the mutant allele frequency in Border Collies is markedly low compared to that in the susceptible dog breeds such as rough and smooth Collies.

Real time PCR for fast detection of the angiotensinogen polymorphisms

to develop a rapid and reliable real-time PCR to detect polymorphisms of angiotensinogen (AGT), to compare the two methods of MS-PCR (Mutagenically Separated PCR) and real-time PCR to determine three polymorphisms of the angiotensinogen gene M235T, the A (-6) G and A (-20) C. the method of real-time PCR was developed on the PLC Roche LightCycler1 with SYBR Green I. We used two sense primers and a primer nonsense. Detection of polymorphisms of angiotensinogen gene was performed by comparing the melting curves. the DNA samples were analyzed by two methods: real-time PCR and MS-PCR. In our study, no differences were found between the two techniques. The real-time PCR is a rapid and reliable method for detecting gene polymorphisms on the AGT M235T, the A (-6) G and A (-20) C. this method of real-time PCR is a reliable genetic test, which is fast and cheap and can be used in practice to study particular polymorphisms of AGT gene associated with cardiovascular disease.

The inflammatory response is influenced by FXIII VAL 34 LEU polymorphism in a human LPS model

In sepsis, activation of coagulation and inhibition of fibrinolysis lead to microvascular thrombosis. The Val34Leu polymorphism affects the function of FXIII by increasing the rate of FXIII activation by thrombin, which results in an increased and faster rate of fibrin stabilization. Sepsis and multi-organ failure cause disturbance of the normal balance of inflammation and coagulation, one of the most frequent causes of death in ICU patients. Research in polymorphism has shown the possible influence of FXIII in coagulation and inflammation. We analyzed the influence of the common FXIII Val34Leu polymorphism on inflammatory and coagulation parameters in human experimental endotoxinemia. Healthy volunteers (n = 62) received 2 ng endotoxin (LPS) per kg body weight as a bolus infusion over 2 min. We developed a new mutagenic separated PCR assay for determination of the FXIII promoter polymorphism. FXIII levels were higher for homozygous carriers of the FXIII polymorphism in comparison with wild-type 34 Val/Val and heterozygous 34 Val/Leu. Interestingly, persons homozygous for the FXIII Val34Leu polymorphism had lower monocyte and neutrophil counts throughout the observation period, yet prothrombin fragment 1+2 and D-dimer levels did not differ after LPS challenge. Our findings indicate that the common FXIII Val34Leu polymorphism is associated with differences in monocyte and neutrophil cell counts in response to systemic LPS infusion in humans.

Association of the polymorphisms of sodium transport related genes with essential hypertension

To investigate the association of the polymorphisms of rs4961 in alpha-adducin (ADD1) and rs28933400 in Na+/K+ -ATPase a2 (ATP1A2) genes, the products of which are important for sodium transport, with essential hypertension. Mutagenically separated PCR (MS-PCR) was used to detect the genotypes of the two loci. The subjects were recruited randomly including 196 patients of essential hypertension and 192 healthy controls. The frequencies of genotypes and alleles of in the ADD1 gene were significantly different between the patients and controls respectively (P=0.03, P=0.04). There was significant relationship between the genotypes of rs4961 and systolic blood pressure and blood sodium concentration. However, there was no significant relationship between the rs4961 genotypes and diastolic blood pressure, body mass index, blood kalium and chlorine concentrations. There was no polymorphism at the rs28933400 locus in the subjects analyzed. The rs4961 polymorphism of the ADD1 gene is associated with essential hypertension, but the rs28933400 locus in the ATP1A2 gene may have no association with essential hypertension in the studied population.

Multiplex Mutagenically Separated Polymerase Chain Reaction Assay for Rapid Detection of Human Mitochondrial DNA Variations in Coding Area

Recently, it has been recognized that information in the mitochondrial DNA (mtDNA) coding region can provide additional forensic discrimination with respect to the standard typing of the D-loop region, increasing the forensic power of mtDNA testing, which is sometimes rather limited. In the present study, we simultaneously typed ten single nucleotide polymorphisms (SNP) in the coding region by use of mutagenically separated polymerase chain reaction (MS-PCR) in the Chinese Chengdu population. This technique, in which different-size allele-specific primers were used, specifically amplified both alleles of mtDNA in the same reaction. Subsequent gel electrophoresis showed ten of the allelic products of different loci. Using multiplex MS-PCR, 30 primers were added simultaneously into one reaction tube to identify ten SNPs. The mtDNA variations of 160 individuals from the Chinese Chengdu population were examined and classified into 18 haplotypes. The multiplex MS-PCR method is suitable for large-scale screening studies of mtDNA variability because it is both rapid and economical.

An Efficient Tool for Surveying CRF01_AE HIV Type 1 Resistance in Thailand to Combined Stavudine–Lamivudine–Nevirapine Treatment: Mutagenically Separated PCR Targeting M184I/V

Under programs organized by the government of Thailand, HIV-1-infected patients have been treated since 2002 with several regimens, including a tablet known as GPOvir, which contains lamivudine, stavudine, and nevirapine. The aim of this study was to establish an effective assay, based on mutagenically separated PCR (MS-PCR), with the goal of surveying GPOvir-resistant HIV-1 cases. To determine the target mutation point for the assay, we analyzed the patterns of acquired drug resistance in plasma samples from GPOvir-failed cases. Of 428 HIV-1-infected individuals treated with GPOvir at Lampang Hospital in northern Thailand from 2002 to 2004, 66 had detectable viral loads after 3 months of treatment. The HIV-1 sequences of these 66 GPOvir-failed cases and 55 pre-GPOvir baseline samples were analyzed. The most prevalent drug resistance mutation among the samples was the lamivudine resistance M184I/V mutation. Based on this finding, we developed a new MS-PCR assay to detect the M184I/V mutation, and evaluated the assay performance for detecting GPOvir-resistant CRF01_AE cases. Comparing the results of M184I/V MS-PCR and sequence analyses, we found a concordance rate of 95% and an overall sensitivity of the M184I/V MS-PCR for detecting GPOvir-resistant cases of 79%. Considering the relatively low price of the assay, approximately $12.50 per sample, M184I/V MS-PCR may be a candidate for monitoring a large number of GPOvir-treated patients, particularly in developing nations.

Prevalence of Cholesteryl Ester Storage Disease

Cholesteryl ester storage disease (CESD) is an autosomal recessive chronic liver disease caused by lysosomal acid lipase (LAL) deficiency. The gene is located on chromosome 10q23.2-q23.3, and the enzyme is essential for triglycerides and cholesteryl ester hydrolysis in lysosomes. CESD is characterized by hypercholesterolemia, hypertriglyceridemia, HDL deficiency, and abnormal lipid deposition in many organs. In the liver this results in hepatomegaly caused by hepatic steatosis and fibrosis that can lead to micronodular cirrhosis.1 Disease onset takes place during childhood or adolescence. Males and females are affected in about equal numbers. Patients rarely reach the age of 30. Biochemically, the disorder is recognized by largely reduced lysosomal acid lipase activity.2,3 Complete absence of LAL activity causes Wolman Disease, which is normally fatal within the first 6 months of life.1,4 Several groups have identified mutations in the LAL gene underlying CESD and Wolman disease.5–9 Mutations causing Wolman disease produce an enzyme with no residual activity or no enzyme at all, whereas CESD-causing mutations encode for LAL which retains some enzyme activity.4,10 A G-to-A transition at position −1 of the exon 8 splice donor (E8SJM, E xon 8 S plice J unction M utation) leads to an in-frame deletion of exon 8. The resulting protein is 24 amino acids shorter and has no residual LAL activity, however E8SJM does not cause Wolman Disease because 2% to 4% of normally spliced LAL is present in homozygote carriers.11,12 The vast majority of CESD …