Multiplex Amplification Refractory Mutation System Research Articles

Characterization of Hemoglobin Malay Phenotypes in Tertiary Hospitals

Hemoglobin (Hb) Malay is a common β hemoglobinopathy in Malaysia caused by A > G mutation in codon 19 leading to β+-thalassemia phenotype. However, screening for Hb Malay is challenging as it is undetectable by routine capillary electrophoresis (CE) or high-performance liquid chromatograpy (HPLC) methods. This study aimed to determine the Hb Malay phenotypes. The study was done on 521 cases with presumed β thalassemia from UKMMC and Hospital Selayang as well as confirmed Hb Malay cases from Hospital Sultanah Bahiyah, Kedah in over a 5-year period. Hb analysis using CE or HPLC followed by multiplex amplification refractory mutation system polymerase chain reaction and DNA sequencing were performed. Significant differences in mean values of haematological parameters among Hb Malay carriers against β thalassemia carriers were determined using one-way ANOVA and ROC analysis. A total of 482/521 cases of β globin mutations were identified. Among these, 54 Hb Malay cases were identified whereby 21 Hb Malay cases were from UKMMC and Hospital Selayang whilst 33 Hb Malay cases were from Hospital Sultanah Bahiyah, Kedah. Fifty-two were Hb Malay carriers whereas two cases were compound heterozygotes. The mean hemoglobin, mean cell volume, mean cell hemoglobin, and HbA of Hb Malay carriers were significantly higher than β° thalassemia carriers. The HbA2 range of Hb Malay carriers was wider (3.5–5.5%) with median value of 3.9%. A new HbA2 cutoff value ≤4.6% (AUC 0.717, p < 0.001) was proposed. Compound heterozygous Hb Malay/IVS1-5(G > C) showed transfusion-dependent thalassemia phenotype. Hb Malay carriers have different red cell and electrophoretic parameters than classical β° thalassemia carriers with wider HbA2 range. HbA2 of ≤4.6% should prompt a molecular confirmation for Hb Malay carrier status.

Hematological Profile of Hb Adana Among High School Students in Northeast Peninsular Malaysia.

Background Hb Adana is a non-deletional alpha (α)-thalassaemia variant resulting from mutations in α1- or α2-globin codon 59 (αCD59), leading to the production of unstable α-globin. Clinical manifestations can vary from silent carrier status to dependence on blood transfusions, hepatosplenomegaly, skeletal deformities, and spinal cord compression. Despite the significance of Hb Adana inheritance, studying this variant poses challenges due to the scarcity of molecular tests and the potential for routine diagnoses to be overlooked. This study aims to investigate the prevalence of Hb Adana among local high school students and assess the hematological parameters and hemoglobin analysis of Hb Adana in Malaysia. Methodology This retrospective study analyzed 13,721 blood samples collected from high school students participating in Malaysia's National Thalassaemia Screening Program at Hospital Raja Perempuan Zainab II (HRPZ II). Deletional α-thalassaemia was detected using multiplex gap-polymerase chain reaction (PCR), while common non-deletional α-thalassaemia was identified using multiplex amplification refractory mutation system (ARMS) PCR. Data were extracted from the HRPZ II database for analysis. Results Among the participants, 2327 individuals were found to have either common deletional (n=1037, 44.6%) or non-deletional (n=1290, 55.4%) α-thalassaemia. Hb Constant Spring was the most prevalent non-deletional α-thalassaemia, accounting for 53.03% of cases. Thirty-one participants (1.33%) exhibited αCD59α/αα, and one (0.04%) had αCD59α/-α3.7. Among the 32 subjects with Hb Adana, 87.5% were Malay, and 12.5% were Orang Asli. Additionally, seven cases of HbE/Hb Adana co-inheritance were identified. Hemoglobin levels in heterozygous Hb Adana individuals ranged from mild anemia to normal, between 95 g/L and 153 g/L. Mean corpuscular volume (MCV) and mean corpuscular hemoglobin (MCH) were approximately 73 fL and 23 pg, respectively. Conclusion This study delineates the distribution ofα-thalassaemia mutation patterns among high school students in Kelantan, Northeast Peninsular Malaysia. Our findings indicate that Hb Adana is rare in our region and co-inheritance with an α-gene deletion results in α+-thalassaemia and with HbE, α0-thalassaemia. All heterozygous Hb Adana individuals exhibited low MCVs and MCHs.

Clinical and haematological characteristics of 38 individuals with Hb G-Makassar in Malaysia.

Haemoglobin (Hb) G-Makassar is a rare Hb variant. It presents a diagnostic challenge as it imitates sickle Hb (Hb S) in standard electrophoresis and high-performance liquid chromatography assays requiring DNA analysis to confirm diagnosis. Both have point mutations in codon 6, exon 1 in the β-globin (HBB) gene with different pathogenicities. This study describes the clinical phenotype, haematology and genotype of Hb G-Makassar. Clinical and laboratory data of 38 cases of Hb G-Makassar over 8 years were analysed. Hb G-Makassar was confirmed by a direct sequencing of HBB gene and co-inheritance of α-thalassaemia determined through multiplex gap-PCR and multiplex Amplification Refractory Mutation System polymerase chain reaction. All cases were Malays, predominantly from Terengganu (n=20, 52.6%). There were 14 (36.8%) males and 24 (63.2%) females with median age of 25 years. Majority (n=33, 86.8%) had features of thalassaemia trait with mean±SD for Hb, mean cell volume (MCV) and mean cell haemoglobin (MCH) as 13.21g/dL±1.69, 73.06±4.48fL and 24.71±1.82pg, respectively. None had evidence of haemolysis or thromboembolic complications. Six genotypes were identified; ßG-Makassar/ß,αα/αα (n=19, 50.0%), ßG-Makassar/ßE,αα/αα (n=4, 10.5%), ßG-Makassar/ßNewYork,αα/αα (n=1, 2.6%), ßG-Makassar/ß,αα/-α (n=11, 28.9%), ßG-Makassar/ß,αα/αAdanaα (n=2, 5.3%) and ßG-Makassar/ß,αα/-SEA (n=1, 2.6%). The ßG-Makassar/ß,αα/αα showed that features of thalassaemia trait with mean±SD for Hb, MCV and MCH were 13.74g/dL±2.40, 76.18±6.02fL and 25.79±2.41pg, respectively. This is the largest study reporting a significant number of Hb G-Makassar in Malaysia. Although the mutation is similar to Hb S, the phenotype is benign.

The use of multiplex ARMS-PCR for mutational analysis of beta-globin gene in consanguineous population of KP Pakistan

Purposeß-thalassemia is a hereditary disorder due to mutation in the ß-globin gene on chromosome 11. Out of 200 known ß-globin gene chain mutations recognized, it is better to identify the most common mutation in specific regions and ethnicity for cost-effective molecular diagnosis of this disorder. Therefore, this study aims to practice multiplex-amplification refractory mutation system (ARMS) PCR on patients with thalassemia in Khyber Pakhtunkhwa (KP) to investigate the most common mutations in the ß-globin chain gene.Design/methodology/approachTwenty-two individuals (patients, their parents and non-affected siblings) with signed consent were studied from six consanguineous families of ß-thalassemia. Blood samples were collected for DNA isolation. For the detection of mutations in the ß-globin gene, ARMS-PCR was used. The amplicon was visualized through 2% Agarose Gel.FindingsThe most common mutations among different ethnic groups in the study area residents were Fr 8-9 (+G) and IVS 1-5 (G&gt; C). The prominent enhancing factors for ß-thalassemia are inter-family marriages and lack of awareness.Practical implicationsMultiplex ARMS_PCR is the most valuable technique for assessing multiple mutations in a single reaction tube.Social implicationsDue to extensively found ethnic and regional variations and a high rate of consanguinity, the Pashtun population has a great risk of mutations in their genome. Therefore, ARMS-PCR is a cost-effective mutational diagnostic strategy that can help to control disease burden.Originality/valueLimited studies using ARMS-PCR for mutational analysis in the ß-globin gene are conducted. This study is unique as it targeted consanguineous families of KP Pakistan.

HBB Gene Mutations and Their Pathological Impacts on HbE/β-Thalassaemia in Kuala Terengganu, Malaysia.

β-thalassaemia is a disorder caused by mutations in the β-globin gene, leading to defective production of haemoglobins (Hb) and red blood cells (RBCs). It is characterised by anaemia, ineffective erythropoiesis, and iron overload. Patients with severe β-thalassaemia require lifelong blood transfusions. Haemoglobin E beta-thalassaemia (HbE/β-thalassaemia) is a severe form of β-thalassaemia in Asian countries. More than 200 alleles have been recognised in the β-globin region. Different geographical regions show different frequencies of allelic characteristics. In this study, the spectrum of β-thalassaemia (β-thal) alleles and their correlation with iron overload, in HbE/β-thalassaemia patients, β-thalassaemia trait, and HbE trait were studied. Blood samples (n = 260) were collected from 65 β-thalassaemia patients, 65 parents (fathers and/or mothers) and 130 healthy control individuals. Haematological analyses, iron profiles, and serum hepcidin levels were examined for all participants. DNA was extracted from patients' and their parents' blood samples, then subjected to PCR amplification. Multiplex amplification refractory mutation system PCR (MARMS-PCR) was conducted for eighteen primers to detect the mutations. There was severe anaemia present in HbE/β-thalassaemia patients compared to their parents and healthy controls. The ferritin and iron levels were significantly increased in patients compared to their parents and healthy controls (p = 0.001). Two common mutations were detected among the patient group and three mutations were detected among their parents, in addition to seven novel mutations in HbE/β-thalassaemia patients (explained in results). Some mutations were associated with severe anaemia in β-thalassaemia patients. The detection of mutations is a prognostic marker, and could enhance the appropriate management protocols and improve the haematological and biochemical statuses of β-thalassaemia patients.

Frequency of Hereditary Hemochromatosis Gene (HFE) Variants in Sri Lankan Transfusion-Dependent Beta-Thalassemia Patients and Their Association With the Serum Ferritin Level.

IntroductionCo-inheritance of hereditary hemochromatosis (HFE) gene variants p. C282Y and p.H63D worsen iron overload in transfusion-dependent thalassemia. Data on the HFE gene variants in Sri Lankan patients with thalassemia have not been extensively studied. This study aimed to analyze the p.C282Y and p.H63D variants in transfusion-dependent beta (β) and HbE/β-thalassemia patients and establish an association between these variants and their serum ferritin levels.Materials and MethodsA total of 125 transfusion-dependent β-thalassemia major and HbE/β thalassemia patients were tested for the c.845G>A (p.C282Y) and c.187C>G (p.H63D) HFE gene variants using the multiplex Amplification Refractory Mutation System Polymerase Chain Reaction method. For phenotype-genotype correlation, serum ferritin levels, the erythrocyte sedimentation rate (ESR), and C-reactive protein (CRP) levels were measured. The standard descriptive statistics were used for data analysis.ResultsThe study cohort consisted of transfusion-dependent 123 β-thalassemia and 2 HbE/β-thalassemia patients. The p.C282Y variant was not detected in any patient; allele frequency for the wild type (c.845GG) was 100%. Twenty-three patients were heterozygous for the p.H63D variant allele, and the allele frequencies were c.187CC 91.8%, c.187CG 9.2%, and c.187GG 0%. The mean serum ferritin level was relatively higher (mean level 4,987 ng/ml) in the p.H63D heterozygous (c.187CG) group compared to the wild type (c.187CC) group (mean level 4,571 ng/ml), but the difference was statistically not significant (p = 0.865). Among the total study population, CRP, ESR, and serum glutamine aspartate transaminase (SGPT) were elevated in 9 (7.2%), 65 (52%), and 82 (65.6%) patients, respectively. Among the p.H63D c.187CG group, elevated CRP, ESR, and SGPT were present in 5 (5%), 15 (12%), and 18 (14.4%) patients, respectively. The detected sample number was low to correlate with the confounding effect of inflammatory disorders and liver damage on the serum ferritin levels.ConclusionsThe HFE gene variant p.C282Y is unlikely to cause iron overload in the Asian β-thalassemia patients; the rarity of this variant in the study cohort replicates the findings of other South Asian population studies of this variant. The presence of the p.H63D variant could be a potential risk factor for iron overload in the β-thalassemia patients. A more extensive cohort study is required to validate this finding.

Detection of Common β-Globin Gene Mutation in Eastern Nepal by Multiplex Amplification Refractory Mutation System Polymerase Chain Reaction System

β-thalassemia is the most common autosomal recessive haemoglobin disorder worldwide. Although more than 200 β-thalassemia mutations have been reported, few ethnic group-specific alleles account for 90-93% of the β-thalassemia alleles in each population. The primary purpose of the study was to identify the prevalence of different types of beta-thalassemia mutations in the study group, and to establish a genotype-phenotype correlation. Complete blood count, peripheral blood smeacr, capillary electrophoresis and multiples arms refractory mutation system based polymerase chain reaction (MARMS) were performed on the peripheral blood samples to detect beta mutations at Decode Genomics and Research Centre, Sinamangal Kathmandu. The MARMS-PCR technique was applied for the detection of nine common mutations IVS1- 5(G&gt;C), 619 bp del, FS8/9 (+G), IVS1-1 (G&gt;T), FS 41/42 (-CTTT), C-15 (G&gt;A). FS 16 (-C), C-30 (G&gt;C) and C-5 (-CTCT). In the study group, three mutations, C-15(G&gt;A), C-26 (Glu-Lys) and IVS1-5 (G&gt;C) were detected. The study revealed a characteristic mutational profile in the five ethnic groups of eastern Nepal. It is the first report of HbE in the Koch Rajbanshi of eastern Nepal. The mutation C-15 reported as rare by earlier workers was the most common mutation identified in this research work.

Four Variations Detected in 3 Genes Related to Thrombophilia by Multiplex ARMS – PCR

Thrombophilia is a condition that may cause venous obstruction, leading to serious illnesses such as stroke, respiratory failure, and even death. This condition is hereditary or related to the immune system. Inherited thrombophilia is screened by analysing genetic variants (allele). In this study, we analyse 4 variations in 3 genes, which occur at high variable frequency, namely Methytetrahydrofolate Reductase (MTHFR C677T, MTHFR A1298C), Factor V Leiden (FVL C1691T) and Plasminogen Activator Inhibitor 1 (PAI-1 5G/4G), using allele-specific polymerase chain reaction (ARMS-PCR). The primers were designed using FastPCR program and PCR conditions were optimized to identify 8 alleles of 4 variants through 3 multiplex ARMS - PCR reactions. The accuracy of the ARMS - PCR results was confirmed by Sanger sequencing and the real time PCR commercial kit (SNP). Mastering the allele specific PCR technique contributes to the survey of genetic variations in the Vietnamese population in order to screen and treat thrombophilia-related diseases.&#x0D; Keywords&#x0D; Thrombophilia, allele-specific polymerase chain reaction (ARMS-PCR), variants MTHFR C677T, MTHFR A1298C, FVL C1691T, PAI-1 4G/5G.&#x0D; References&#x0D; [1] A. Dautaj, G. Krasi, V. Bushati et al., Hereditary Thrombophilia, Acta Biomedica, Vol. 90, No. 10S, 2019, pp. 44-46, https://doi.org/10.23750/abm.v90i10-S.8758.[2] M. G. Beckman, W. C. Hooper, S. E. Critchley, T. L. Ortel, Venous Thromboembolism: A Public Health Concern, American Journal of Preventive Medicine, Vol. 38, No. 4S, 2010, pp. S495-S501, https://doi.org/10.1016/j.amepre.2009.12.017.[3] G. E. Raskob, R. Silverstein, D. W. Bratzler et al., Surveillance for Deep Vein Thrombosis and Pulmonary Embolism: Recommendations from A National Workshop, American Journal Preventive Medicine, Vol. 38, No. 4S, 2010, pp. S502-S509, https://doi.org/10.1016/j.amepre.2010.01.010.[4] J. A. Heit, M. D. Silverstein, D. N. Mohr et al., The Epidemiology of Venous Thromboembolism in the Community, Thrombosis and Haemostasis, Vol. 86, No. 1, 2001, pp. 452-463, https://doi.org/10.1055/s-0037-1616243.[5] M. Cushman, A. W. Tsai, R. H. White et al., Deep Vein Thrombosis and Pulmonary Embolism in Two Cohorts: The Longitudinal Investigation of Thromboembolism Etiology, The American Journal of Medicine, Vol. 117, No. 1, 2004, pp. 19-25, https://doi.org/10.1016/j.amjmed.2004.01.018.[6] J. A. Heit, The Epidemiology of Venous Thromboembolism in the Community: Implications for Prevention and Management, Journal of Thrombosis and Thrombolysis, Vol. 21, No. 1, 2006, pp. 23-29, https://doi.org/10.1007/s11239-006-5572-y.[7] R. H. White, The Epidemiology of Venous Thromboembolism, Circulation, Vol. 107, No. 23 S1, 2003, pp. I4-I8, https://doi.org/10.1161/01.cir. 0000078468.11849.66.[8] A. Wahed, A. Dasgupta, Thrombophilias and Their Detection, Hematology and Coagulation: A Comprehensive Review for Board Preparation, Certification and Clinical Practice, Elsevier, United States of America, 2015, pp. 263-275.[9] SNP Biotechnology, SNP Detection Real-Time PCR Kits, http://www.snp.com.tr/EN,37/snp-detection-real-time-pcr-kits.html/, 2021 (accessed on: May 1st, 2021).[10] V. S. Le, K. T. Tran, H. T. P. Bui et al., A Vietnamese Human Genetic Variation Database, Human Mutation, Vol. 40, No. 10, 2019,pp. 1664-1675, https://doi.org/10.1002/humu.23835.[11] dbSNP – NCBI, Reference SNP (rs) Report (rs6025, rs1799762), https://www.ncbi.nlm.nih.gov/snp/,02021 2021 (accessed on: May 1st, 2021).[12] D. C. Rees, M. Cox, J. B. Clegg, World Distribution of Factor V Leiden, The Lancet, Vol. 346, No. 8983, 1995, pp. 1133-1134, https://doi.org/10.1016/s0140-6736(95)91803-5. A. Fateh, M. Aghasadeghi, S. D. Siadat et al., Comparison of Three Different Methods for Detection of IL28 rs12979860 Polymorphisms as A Predictor of Treatment Outcome in Patients with Hepatitis C Virus, Osong Public Health and Research Perspectives, Vol. 7, No. 2, 2016, pp. 83-89, https://doi.org/10.1016/j.phrp.2015.11.004. [13] T. Huang, J. Zhuge, W. W. Zhang, Sensitive Detection of BRAF V600E Mutation by Amplification Refractory Mutation System (ARMS)-PCR, Biomarker Research, Vol. 1, No. 3, 2013, https://doi.org/10.1186/2050-7771-1-3.[14] R. F. V. Medrano, C. A. D. Oliveira, Guidelines for the Tetra-primer ARMS-PCR Technique Development, Molecular Biotechnology, Vol. 56, 2014, pp. 599-608, https://doi.org/10.1007/s12033-014-9734-4.[15] C. Runcharoen, K. Fukunaga, I. Sensorn et al., Prevalence of Pharmacogenomic Variants in 100 Pharmacogenes Among Southeast Asian Populations under Tarmacogenomics Research Network (SEAPharm), Human Genome Variation, Vol. 8, No. 7, 2021, https://doi.org/10.1038/s41439-021-00135-z.

Molecular Evaluation of Chlamydia trachomatis Infection and Its Association with Tumor Necrosis Factor-α Polymorphism in Recurrent Spontaneous Abortions

The rate of infections in the intensive care units (ICUs) is rising, mainly because of the increasing use of invasive procedures. Several bacterial infections and host genetic backgrounds, including TNFα SNP polymorphisms, play important roles in recurrent spontaneous abortions (RSA). So, this study aimed to evaluate C. trachomatis infection and its relation with TNFα -308 and -238 polymorphism for early detection and treatment of RSA. The blood samples were taken from 63 Iranian women with a history of RSA and 59 ethnically matched healthy controls with at least two successful pregnancies and no history of abortion. Then, DNA was extracted from all samples and detection of C. trachomatis and TNFα -308 and -238 polymorphism was determined by multiplex amplification-refractory mutation system (ARMS)-PCR. Finally, the statistical analysis to detection C. trachomatis and host genetic roles in RSA were analyzed by Epi Info TM software by X2 test. C. trachomatis was detected in 22 and 3% of the RSA and the control group, respectively. Moreover, in comparison with healthy controls, C. trachomatis infection was closely correlated with TNFα -308 genotypes, whereas no significant association was observed between TNFα -238G/A and RSA. In addition, statistical analysis of TNFα -308 genotypes showed that the frequency of genotype AA was higher in patients with C. trachomatis infections than healthy individuals and the difference was statistically significant. This study demonstrated that molecular analysis of TNF -308 genotypes is important in early detection and treatment of RSA with C. trachomatis infection.

Association study of interleukin-1 receptor associated kinase 1 rs3027898 A/C gene polymorphism and preeclampsia in Pakistani population.

To find the association between interleukin-1 receptor-associated kinase 1 rs3027898 gene polymorphism and preeclampsia. The case-control study was conducted from October, 2018 to September, 2019 at the Railway General Hospital and the Department of Biochemistry, Islamic International Medical College, Rawalpindi, Pakistan, and comprised patients diagnosed with preeclampsia and healthy controls. The interleukin receptor-associated kinase-1 polymorphism was determined using multiplex tetra primer amplification refractory mutation system polymerase chain reaction. Outcomes were determined in terms of association of interleukin receptor-associated kinase-1 with preeclampsia. Data was analysed using SPSS 22. Of the 160 subjects, 80(50% were cases with a mean age of 30±5.3 years and 80(50%) were controls with a mean age of 27±3.7 years. AC genotype was seen in 45(56.25%) cases and 30(37.5%) controls, AA genotype in 25(31.25%) cases and 30(37.5%) controls, while CC genotype was seen in 10(12.5%) cases and 20(25%) controls (p>0.05). There was no significant association of interleukin receptor-associated kinase-1 genotypes with preeclampsia.

Prevalence and distribution of major β-thalassemia mutations and HbE/β-thalassemia variant in Nepalese ethnic groups

BackgroundBeta-thalassemia is a genetic disorder that is inherited in an autosomal recessive pattern. This genetic disease leads to a defective beta-globin hemoglobin chain causing partial or complete beta-globin chain synthesis loss. Beta-thalassemia major patients need a continuous blood transfusion and iron chelation to maintain the normal homeostasis of red blood cells (RBCs) and other systems in the body. Patients also require treatment procedures that are costly and tedious, resulting in a serious health burden for developing nations such as Nepal. MethodsA total of 61 individuals clinically diagnosed to have thalassemia were genotyped with multiplex amplification refractory mutation system–polymerase chain reaction (ARMS–PCR). Twenty-one major mutations were investigated using allele-specific primers grouped into six different panels. ResultsThe most common mutations found (23%) were IVS 1–5 (G-C) and Cd 26 (G-A) (HbE), followed by 619 deletion, Cd 8/9 (+G), Cd 16 (–C), Cd 41/42 (–TTCT), IVS 1–1 (G–T), Cd 19 (A-G), and Cd 17 (A-T) at 20%, 12%, 8%, 6%, 4%, 3%, and 1%, respectively. ConclusionThe results of this study revealed that Nepal's mutational profile is comparable to that of its neighboring countries, such as India and Myanmar. This study also showed that thalassemia could be detected across 17 Nepal's ethnic groups, especially those whose ancestors originated from India and Central Asia.

Association between apolipoprotein E polymorphism and lipid profile in patients of myocardial infarction

Objective: To determine the effect of apo E polymorphism on lipid profile in patients of myocardial infarction as well as normal healthy controls. Subjects and Method: Total 100 acute myocardial infarction patients with age and gender matched controls, within age ranging from 25 to 80 years were included. Lipid profile levels of MI patients and controls were estimated by standard methods. DNA’s were extracted by salting out method and Genotypes for Apo-E were determined by Multiplex Amplification Refractory Mutation System PCR. Results: The total cholesterol, LDL cholesterol, TC/HDL-C ratio, LDL-C/HDL-C ratio level was significantly increased (p<0.01) in E4E4 allele than E3E3 allele. Analysis of variants has significant difference (p<0.01) observed in total cholesterol and LDL cholesterol levels in all apo E alleles of MI patients. Conclusion: Our results suggestive that the risk of myocardial infarction associated with apo E4E4 alleles which are major risk factor for developing the coronary artery disease.

Response of Patients with Transfusion-Dependent β-Thalassemia (TDT) to Betibeglogene Autotemcel (beti-cel; LentiGlobin for β-Thalassemia) Gene Therapy Based on HBB Genotype and Disease Genetic Modifiers

Response of Patients with Transfusion-Dependent β-Thalassemia (TDT) to Betibeglogene Autotemcel (beti-cel; LentiGlobin for β-Thalassemia) Gene Therapy Based on HBB Genotype and Disease Genetic Modifiers

Associations Between the Apolipoprotein E ε4 Allele and Reduced Serum Levels of High Density Lipoprotein a Cognitively Normal Aging Han Chinese Population.

Background: Previous studies have confirmed that APOE genotype is associated with lipid metabolism, but related studies are inconsistent. Therefore, we conducted this cross-sectional study to explore the associations between apolipoprotein E (APOE) genotypes and serum levels of fasting blood sugar, triglycerides, total cholesterol, high density lipoprotein, and low density lipoprotein in a cognitively normal aging Han Chinese population.Methods: One hundred sixty-nine community elders with normal cognitive function were included in the study. Based on multiplex amplification refractory mutation system polymerase chain reaction (PCR), these subjects were divided into three groups: (1) E2/2 or E2/3 (APOE E2); (2) E3/3 (APOE E3); and (3) E2/4, E3/4, or E4/4 (APOE E4). Correlations of serum levels of fasting blood sugar, triglycerides, total cholesterol, high density lipoprotein, and low density lipoprotein with APOE genotypes were assessed.Results: The results of Mann-Whitney analysis showed that the concentration of high density lipoprotein (HDL) in APOE E2 and E3 groups was higher than that in E4 groups (p < 0.05). Logistic regression analysis also suggested that a lower level of high density lipoprotein was associated with the E4 allele (adjusted odds ratio 0.164, 95% confidence interval 0.031~ 0.876, P = 0.034).Conclusion: APOE E4 is associated with decreased serum high density lipoprotein concentration in healthy elderly. However, the above conclusions need to be further verified.

Iranian patients with hemoglobin H disease: genotype-phenotype correlation.

Thalassemia is one of the most common monogenic hereditary disorders. Despite noticeable advances made in prevention strategies, it is still highly prevalent in the Iranian population. A key approach to management and early diagnosis of the disease is through revealing the regions with high prevalence and determining common genetic and phenotypic diversity. In the current study Hemoglobin H (HbH) disease patients were analyzed as the most common form of thalassemia intermedia in Iran. A total of 80 patients suspected of being thalassemic according to their mild to moderate anemia, microcytosis and normal iron levels were included in this study at the hemoglobinopathy and thalassemia center of Ahvaz University of Medical Science. Patients were analyzed for hematological parameters and HbH mutations using Multiplex Gap Polymerase Chain Reaction and Multiplex Amplification Refractory Mutation System. Twelve mutations were detected in the studied population. The most common genotype was -α3.7/--MED (45%) followed by Homozygote αPoly A2 (17.5%). A total of ten different alpha-globin (α-globin) mutations were observed in patients which --MED, being the most common mutation (26.27%), followed by -α3.7 (24.37%) and αpolyA2(A>G) (18.12%). Hematological parameters such as Hb, MCV, MCH and HbH were assessed and results showed that they varied significantly among genotypes, adjusted to age and gender. This study reveals a highly diverse range of HbH patients different from what was thought in terms of both genotype and phenotype in the Khuzestan region of Iran. These findings could contribute to improve the thalassemia managing policies in this province.

Prevalence and Genetic Analysis of α- and β-Thalassemia and Sickle Cell Anemia in Southwest Iran

This prospective study assessed the prevalence and genetic analysis of α- and β-thalassemia and sickle cell anemia (SCA) in Southwest Iran. Hematological indices were measured in 17,581 couples living in Khuzestan Province, Southwest Iran. Individuals with mean corpuscular volume <80, mean corpuscular hemoglobin <27, hemoglobin A2 ≥3/5 were considered as β-thalassemia traits. Prevalence of minor β-thalassemia, α-thalassemia, SCA, iron deficiency anemia, and silent thalassemia were respectively identified in 995 (5.6%), 1169 (6.65%), 1240 (7.05%), 911 (5.18%), and 1134 (6.45%) individuals using a multiplex amplification refractory mutation system, and direct DNA sequencing of globin genes. Three codons IVS-II-1 (G → A; 26%; n = 13), IVS-I-1 (G → T; 16%; n = 8), and IVS-I-110 (G → A; 14%; n = 7) were the most frequent mutants and IVS-II-1 was the most common β-thalassemia mutation. Also, based on a gap-polymerase chain reaction assay, genotype frequencies of α -globin mutations were –α3.7kb (50%; n = 25), Med/ααthal (12%; n = 6), and –α 4.2/αα (10%; n = 5), which were the most frequent deletion mutants (72% in total). The most common deletion (50%) was –αα3.7kb. Our data suggest that the population of Southwest Iran is at high risk of α- and β-thalassemia caused by these deletion mutants and SCA. Our findings will be useful for developing an efficient control program and genetic counseling.

Multiplex amplification refractory mutation system PCR (ARMS-PCR) provides convenient method for differentiation of canine parvovirus vaccine and field strains.

Vaccination with canine parvovirus-2 (CPV-2) modified live attenuated vaccine remains an effective control strategy for preventing parvovirus induced enteritis in dogs. Virus shedding is a common phenomenon few days after vaccination, possessing a diagnostic dilemma for accurate differentiation of CPV-2 vaccine and wild type field strains. Though several molecular approaches are available for differentiation, the present study focuses on a simple, rapid, cost-effective differentiating infected from vaccinated animals strategy employing ARMS-PCR for differentiation of CPV-2 vaccine and wild type field strains. The ARMS-PCR was initially validated using positive controls of recombinant plasmids, further used for screening six commercial CPV-2 vaccines and 24 archived CPV-2 positive field samples as well as to check fecal shedding of vaccine virus for 10days post-vaccination in three vaccinated dogs. Sequencing of randomly selected CPV-2 commercial vaccine strains and archived field samples confirmed authenticity of the developed ARMS-PCR assay.

Genotype-phenotype correlation among beta-thalassemia and beta-thalassemia/HbE disease in Thai children: predictable clinical spectrum using genotypic analysis.

IntroductionBeta-thalassemia is a group of inherited hemolytic anemias and one of the most common genetic disorders in Thailand. The clinical spectrum of beta-thalassemia disease ranges from mild to severe clinical symptoms including mild beta-thalassemia intermedia (TI) and severe beta-thalassemia major (TM).ObjectiveThis study aimed to determine the correlation between beta-globin gene (HBB) mutations and their phenotypic manifestations by evaluating patients’ clinical characteristics, transfusion requirements, growth and hematologic parameters, and hemoglobin typing among pediatric patients treated at Phramongkutklao Hospital.Materials and methodsSeventy beta-thalassemia patients, including 63 with beta-thalassemia/hemoglobin E (HbE) and 7 with either homozygous or compound heterozygous beta-thalassemia, were enrolled in this study. Their clinical presentation, growth parameters and laboratory findings were reviewed and analyzed. The mean follow-up time was 10.52±5.62 years. Mutation analysis in each individual was performed using multiplex amplification refractory mutation system (M-ARMS), direct DNA sequencing of beta-globin gene and gap PCR for 3.4 kb deletion detection.ResultsAll 7 homozygous and compound heterozygous beta-thalassemia patients were classified in TM. Among 63 patients with beta-thalassemia/HbE, 58 were classified in TM and 4 were classified in TI. Mean age at diagnosis was 0.8±0.49 years for homozygous or compound heterozygous beta-thalassemia and 3.43±3.5 years for beta-thalassemia/HbE. The most common HBB mutation was HBB:c.126_129delCTTT [codon 41/42 (-TCTT)] found in 34 alleles (48.6%). The height for age was also lower in homozygous beta-thalassemia patients (<3rd percentile) compared to compound heterozygous beta-thalassemia patients (25–50th percentile).ConclusionThis study revealed a genotype–phenotype correlation of the most prevalent beta-thalassemia in Thai children using diagnostic capacity in genotypic analysis of HBB mutation. Our findings can provide a better prediction of clinical manifestation and severity by early identification of the type of the HBB mutations.

A cross-sectional study of inpatients with late stage of dementia in Southeast China and the associations between biochemical parameters and apolipoprotein E genotypes.

BackgroundThe purpose of this study was to analyze the disease distribution of patients in the late stage of dementia through a cross-sectional investigation and to clarify the association between apolipoprotein E (APOE) genotypes and the serum levels of total cholesterol, total triglycerides, and blood glucose in the late-stage Alzheimer’s disease (AD) patients.MethodsPatients who were in the late stage of dementia in a mental health center were enrolled in this study. A broad battery of neuropsychological tests and neuroimaging was applied to make the diagnosis. The APOE genotype was determined by the multiplex amplification refractory mutation system polymerase chain reaction. The association between APOE genotype and the blood level of biochemical parameters was studied.ResultsA total of 155 patients were enrolled in this study. The majority of patients had AD (67.8%), followed by vascular dementia (15.5%), mixed dementia (9%), and others (7.7%). The APOE ε4 allele frequency was significantly different in the different groups. The serum level of total cholesterol (TC) in APOE ε4 carriers was higher than in non-carriers (P<0.05). No statistically significant differences were found in the blood glucose and triglycerides (TG) levels between these two groups.ConclusionTo our knowledge, this is the first paper to study the characteristics of late-stage dementia in hospital patients in Southeast China. We found that the disease distribution was quite consistent with previous prevalence studies. Moreover, we found that the serum level of TC was higher in APOE ε4 carriers group. However, no association was found between the APOE ε4 allele and serum levels of glucose, TC and TG in the late stage of AD.

RAPID DETECTION OF -THALASSEMIA MUTATIONS IN THAILAND USING MULTIPLEX ARMS

The number of mutations underlining b-thalassemia generate a wide variety of different clinical phenotypes. An understanding of the genotype is important for medical personnel in order to provide proper counseling to patients and their families. Characterization of these mutations should aid the planning of a prenatal diagnosis program for bthalassemia. The heterogeneity of the mutations makes it difficult and time consuming to identify the mutation in some individuals. We developed a single-tube multiplex amplification refractory mutation system (multiplex ARMS) to identify common ethnic- specific b-thalassemia mutations. Confirmation of multiplex ARMS results was carried out using direct sequencing. Three thousand three hundred twenty two people from Phitsanulok province were screened for the b-thalassemia trait by quantitation of HbA2 with microcolumn chromatography and the genotypes of mutations were characterized using multiplex ARMS and direct sequencing. We found that the deletion at codons 41/42 (-TCTT) was the most frequent (48%), codon 17 (A®T) (30%), -28 (A®G) (6%) and IVS-I-1(G®T) (6%) were the second and third in frequency respectively. A -87 (C®A) mutation (4%), IVS II-654 (C®T) (2%), codons 71/72 (+A) (2%) and codon 35 (C®A) mutations (2%) were also found. These techniques were found to be a valuable tool for analysis of b-thalassemia mutations because they are accurate, simple, and speedy in operation. The application for the diagnosis of severe thalassemia in high-risk pregnancies is promising.