Copy number variation of FCGR3B gene among severe dengue patient in Malaysia / Umi Shakina Haridan

Fc Gamma Receptor 3B (FcyRIIIB, encoded by the gene FCGR3B) plays a crucial role in immunity triggered by cellular effector and regulatory functions. Copy number variation (CNV) of this gene has been previously reported to affect susceptibility to several autoimmune diseases and chronic inflammatory conditions. However, it remains a challenge to accurately determine the copy number of this gene in different individuals. Thus this study aimed to establish the most robust CNV genotyping assay by comparing the accuracy and efficiency of (i) quantative PCR (qPCR), (ii) Sequenom Mass ARRAY, and (iii) Paralogue Ratio Test-Restriction Enzyme Digest Variant Ratio (PRT-REDVR). Subsequently the distribution of FCGR3B CNV among the dengue patients in Malaysia was characterized and its association with the severity of the disease was determined. A total of 237 samples were recruited from various study hospitals, of which 191 samples were included into further experiments. 120 were clinically diagnosed as severe dengue or warning sign, while 71 were dengue fever (DF). In the comparison of the three CNV genotyping assays, qPCR showed a considerably broader distribution of signal intensity compared to the other assays, potentially introducing error in estimation of copy number. Both Sequenom and PRTREDVR showed lesser systematic bias, and estimate copy number within the correct range, although PRT-REDVR appears to be more precise and accurate method when genotype FCGRSB. Collectively PRT-REDVR was considered to be most appropriate in the study of multiallelic CNV of FCGR3B. Multiple independent assays should be considered to accurately genotype the CNV of FCGR3B. In the second part of the study, 168 dengue samples (108 case, defined as dengue patients with signs of vascular leakage, and 60 control, defined as dengue patients without vascular leakage) and 52 of healthy samples genotyped with PRT-REDVR were included in the genetic association study. The analysis revealed statistical significance between CNV of FCGR3B of control, case and dengue sample against CNV of FCGR3B of healthy sample, respectively (p = 0.012, p = 0.007 and p = 0.012). On the other hand, there is no significance association shows between case and control (p = 0.301). However, a trend towards the low copy number (CN <2) in case was observed, hence postulating that lower copy number may be attributed with vascular leakage in dengue. Larger number of samples however, is needed to address this postulation

Copy Number Variation of FCGR3B on Negrito-Mendriq / Mohd Helmy Yusof, Umi Shakina Haridan and Muhamad Rahimi Che Hassan

Fc Gamma Receptor 3B (FcγRIIIB, encoded by the gene FCGR3B) plays a crucial role in immunity response activated by cellular effectors and regulatory functions. Copy number variation (CNV) of this gene has been previously reported to affect susceptibility to several diseases such as autoimmune diseases and chronic inflammatory response. One of the rarest and smallest tribes of Orang Asli which is Negrito-Mendriq sample was used as a subject in this study. Twenty-two (22) Lymphoblastoid cell lines (LCLs) of Negrito-Mendriq was obtained from the deposited cell archive, Institute Medical Molecular Biotechnology, IMMB (UiTM Sg Buloh, Selangor, Malaysia) and the LCLs were revived. The DNA was extracted from the LCL and finally PRT – REDVR assay was carried for FCGR3B copy number genotyping. Analysis revealed that copy number 2 demonstrated the highest distribution for FCGR3B (90.9%); whilst no copy number loss was found for FCGR3B. For high copy number (>2), 9.1% was obtained. Copy number greater than 3 (>3) was not found in this study. Finally, it was concluded that no significant difference of CNV of FCGR3B for Orang Asli Negrito-Mendriq when compared with the reported studies. CNV of FCGR3B genes of Negrito-Mendriq was found normal in this study, where the pattern of copy number distribution was almost similar with previously reported studies

Socio-demographic factors of allergy awareness among pre-university Student in Selangor

Allergic disease is a worldwide public health issues and substantially growing over the past 20 years. Due to drastically changes of environmental and dietary culture within the population, Malaysia as one of the developing country is no exception to face the same problem. This study aimed to assess the awareness of allergy diseases and its prevalence using cross-sectional survey method. This study that has been conducted to 138 students from University Teknologi Mara Cawangan Dengkil, Selangor, Malaysia. A self-administered questionnaire was used as an instrument for data collection. Based on sociodemographic data, the mean of knowledge between science and non-science students showed the significant difference. Respondents from Science course score significantly higher for part B (4.54 ± 1.654) compare with other courses. The outcome of the study also showed that, among 138 respondents, n=80 (58%) of them perceived themselves to suffer allergy diseases and it symptoms. Common food to cause food allergy in this study were identified as seafood with n=43 (31.25%) followed with dust with n=40 (29%), peanuts with n=14 (10.1%). The study also indicated the intricate situation of cross reactivity as n=21(15.2%), n=6 (4.3%) and n=1(0.7) of respondents suffered with two, three and four multi allergy reaction respectively. The factor such as family history which subjected to predisposition of genetic factor was strongly associated with occurrence of allergic among respondents. It determined from Chi Square Test (X²) that showed that, there is high significant association (P<0.001) between parental allergies with respondents allergies. As overall, it can be conclude that the knowledge of allergy term, cause and prevention were relatively low among population under study as compared to the previous study

Self-reported on food allergy among university students in Dengkil, Selangor

Prevalence of allergy is a worldwide issue. The outbreak contributes to social and economic burden of the sufferer as well as the society. In Malaysia, the condition has continued to worsen due to the misconception of certain allergy symptom. It also leads to improper clinical diagnosis which affects the accuracy of treatment and upturns the cost further. This circumstances are corresponded to the level of public awareness and knowledge about the terms, symptoms and allergy prevention. This study highlighted the allergy prevalence among young adults based on self–reported approach. This cross-sectional study involved 138 students between the age of 18 to 19 years old from the Centre of Foundation Studies, UiTM Cawangan Selangor, Kampus Dengkil. Overall, the allergy prevalence reported in this study showed higher occurrence compared to previous study of self-reported and clinical data within the same population. The data indicated that n=80 (58%) of respondents indicating seafood such as shrimp as the most common implicated food allergen at n=43 (31.25%) followed by house dust mite at n=40 (29%) and peanuts at n=14 (10.1%). Pearson correlation between respondents’ knowledge of allergy terms, causes and symptoms with allergy prevention and treatment practice shows high significant correlation with (r = 0.387, P<0.001). Chi Square Test among students with allergies who were getting consultation from health personnel showed significant association (P<0.001) in which 41 (59.4%) of students with allergies do not consult any health personnel. As a conclusion, self-reported is only useful to give surface overview on allergy prevalence, but detailed clinical diagnosis is compulsory to ensure the effectiveness of allergy prevention and treatment in the future

A Comparison of Assays for Accurate Copy Number Measurement of the Low-Affinity Fc Gamma Receptor Genes FCGR3A and FCGR3B

The FCGR3 locus encoding the low affinity activating receptor FcγRIII, plays a vital role in immunity triggered by cellular effector and regulatory functions. Copy number of the genes FCGR3A and FCGR3B has previously been reported to affect susceptibility to several autoimmune diseases and chronic inflammatory conditions. However, such genetic association studies often yield inconsistent results; hence require assays that are robust with low error rate. We investigated the accuracy and efficiency in estimating FCGR3 CNV by comparing Sequenom MassARRAY and paralogue ratio test-restriction enzyme digest variant ratio (RT-REDVR). In addition, since many genetic association studies of FCGR3B CNV were carried out using real-time quantitative PCR, we have also included the evaluation of that method’s performance in estimating the multi-allelic CNV of FCGR3B. The qPCR assay exhibited a considerably broader distribution of signal intensity, potentially introducing error in estimation of copy number and higher false positive rates. Both Sequenom and PRT-REDVR showed lesser systematic bias, but Sequenom skewed towards copy number normal (CN = 2). The discrepancy between Sequenom and PRT-REDVR might be attributed either to batch effects noise in individual measurements. Our study suggests that PRT-REDVR is more robust and accurate in genotyping the CNV of FCGR3, but highlights the needs of multiple independent assays for extensive validation when performing a genetic association study with multi-allelic CNVs

Self-perceive on food allergy prevalence among university student in Selangor

Prevalence of allergy is a worldwide issue. The outbreak contributes to social and economic burden of the sufferer as well as the society. In Malaysia, the condition has continued to worsen due to the misconception of certain allergy symptom such as food allergy versus food intolerance. It also lead to improper clinical diagnosis which effect treatment accuracy and upturn the cost of the treatment. This circumstances are corresponded to the level of public awareness and knowledge about the term, signs and symptom of allergy prevention. This study aimed to highlight the allergy prevalence among young adult based on self–perceive approach. This cross-sectional study involved 138 respondents of students with the age of 18 to 19 years old from Centre of Foundation Study, UiTM Cawangan Selangor, Kampus Dengkil. Overall, the allergy prevalence reported within the study population comparatively to another self-perceive and clinical data from previous study showed the higher occurrence. The data indicated that n=80 (58%) of respondents facing allergy problems indicating seafood such as shrimp as the most common implicated food allergen with n=43 (31.25%) followed with house dust mite with n=40 (29%)and peanuts with n=14 (10.1%). Pearson correlation between respondent’s knowledge of allergy term, cause and symptom with allergy prevention and treatment shows weak highly significant correlation with (r = 0.387, P<0.001). Chi Square Test among students with allergies who were getting consultation with health personnel shows significant association (P<0.001) in which 41 (59.4%) of students with allergies do not consult with health personnel. The study also specified the shortcoming of the self- perceive approach due to the symptom misinterpretation and the quality of the survey instrument. As a conclusion self-perceive only, is useful to give surface overview on allergy prevalence, but detail clinical diagnosis is compulsory to ensure the effectiveness of allergy prevention and treatment in future

Analysis of raw quantification data (logEC50) for Sequenom MassARRAY.

<p>Correlation between raw individual copy number calls from the probes designed at 5′-, 3’-, and centre regions of the genes <i>FCGR3A</i> (A, B, and C) and <i>FCGR3B</i> (D, E, and F). Note that there is a clear seperation of 2 cluster plots indicating batch effects, as circled, due to different experimental batches. <b>A</b>, <b>3’ vs 5′; B, 3’ vs centre;</b> and <b>C, 5′ vs centre</b>; <b>D</b>, <b>5′ vs 3’; E, 3’ vs centre;</b> and <b>F, 5′ vs centre.</b> A and D, correlation between the 5- and 3- prime probes were slightly affected, probably due to the probes distance.</p

Percentage of concordant results of <i>FCGR3B</i> CN calls between the three different assays.

<p>Percentage of concordant results of <i>FCGR3B</i> CN calls between the three different assays.</p

Primer sequences and single nucleotide variation (SNV) identified for <i>FCGR3A/B</i> in Sequenom MassARRAY.

<p>Primer sequences and single nucleotide variation (SNV) identified for <i>FCGR3A/B</i> in Sequenom MassARRAY.</p

Scatterplot showing all 99 samples matched for <i>FCGR3B</i> CNV with PRT-REDVR, Sequenom and qPCR.

<p><b>A.</b> qPCR vs Sequenom. qPCR values represent raw copy number, Sequenom values represent absolute copy numbers, <b>B.</b> Sequenom vs qPCR. Both Sequenom and qPCR raw copy number values are presented, showing a low concordance rate between the two platforms, <b>C.</b> qPCR vs PRT-REDVR. qPCR values represent raw unrounded copy number, PRT-REDVR values represent absolute copy number; and <b>D.</b> Sequenom vs PRT-REDVR. Sequenom values represent raw unrounded copy numbers, PRT-REDVR values represent absolute copy number. The broader scater plots of qPCR signal intensity show wider variability of copy number calls, suggesting more copy number calls from qPCR were discordant with the Sequenom and PRT-REDVR. D exhibits a more clustered plot indicating higher agreement of CN calls between Sequenom and PRT.</p