Optimization of Droplet Digital Polymerase Chain Reaction (Ddpcr) For Dna Copy Number Variation Analysis of Cnv Esv27061 in Young Adults with Higher Blood Pressure

Introduction: Copy number variation (CNV) caused by changes in DNA sequences of 1000 or more bases is implicated with susceptibility to common diseases. A study on CNV esv27061 among hypertensive Australian adults reported association with high blood pressure (BP). In Malaysia, no study on CNV among hypertensive young adults is available. Thus, this investigation aimed to assess the CNV esv27061 of young Malaysian adults with high blood pressure using optimized ddPCR. Materials and method: Ten samples each from hypertensive and healthy controls were randomly selected from samples collected for an on-going comparative cross-sectional research project among young adults living in Kuantan. The DNAs were purified using Maxwell RSC Buffy Coat DNA Kit and the concentration was measured using SimpliNano spectrophotometer. Subsequently, restriction digestion of DNAs by EcoRV was performed prior to ddPCR. The products were later subjected to droplet generation (QX100 Droplet Generator), PCR amplification and finally CNV was read by QX100 Droplet reader. Unfortunately, the above method did not yield any result. Thus, an alternative method in which purified DNA concentration was determined by QuantiFluor ONE dsDNA System (Quantus fluorometer). The DNAs (60 ng) and Alu1 were added in master mix during ddPCR and CNV esv27061 analysis was performed as stated above. Results: Optimization of method in this study showed that the detection of CNV esv27061 was possible by the use of more sensitive measurement of DNA concentration, Alu1 restriction enzyme instead of EcoRV and digestion in ddPCR reaction method rather than prior digestion. The finalized protocol run on selected hypertensive and healthy controls has shown to be reproducible and easily interpretable discrimination of gene's copy numbers. Conclusion: This optimized protocol for CNV esv27061 analysis proved useful in identifying CNV and will allow a reproducible assay evaluation and the application of this method to a bigger sample size.

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Copy number variation of CNVesv27061 analysis among young adults with high blood pressure using optimized droplet digital polymerase chain reaction (ddPCR) method

Materials Today Proceedings ◽

10.1016/j.matpr.2019.06.127 ◽

2019 ◽

Vol 16 ◽

pp. 2313-2318

Author(s):

Siti Radziah S. A. ◽

Azarisman S. M. S. ◽

Aida N. S. M. S. ◽

Norlelawati A. T. ◽

Aszrin A.

Keyword(s):

Blood Pressure ◽

Polymerase Chain Reaction ◽

Young Adults ◽

Copy Number Variation ◽

High Blood Pressure ◽

Copy Number ◽

Chain Reaction ◽

Number Variation ◽

Polymerase Chain ◽

Digital Polymerase Chain Reaction

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The Identification of Copy Number Variation of Cnv Esv27061 among Young Adults with Hypertension: Preliminary Findings

IIUM Medical Journal Malaysia ◽

10.31436/imjm.v16i1.1182 ◽

2017 ◽

Vol 16 (1) ◽

Author(s):

Siti Radziah Shaik Alaudeen ◽

Azarisman Shah Mohd Shah ◽

Norlelawati Abdul Talib ◽

Aszrin Abdullah

Keyword(s):

Young Adults ◽

Copy Number Variation ◽

Dna Sequences ◽

Copy Number ◽

Preliminary Finding ◽

Cross Sectional ◽

Genomic Variants ◽

Study Results ◽

Number Variation ◽

Mild Hypertensive

Introduction: Hypertension related morbidities and mortalities around the world show a gradual increase and early detection and prevention are advocated. The Database of Genomic Variants (DGV) has associated variation in DNA sequences called copy number variation (CNV) with susceptibility to common diseases. However, little is known about CNV role in essential hypertension. Thus, this study aimed to characterize the CNV esv27061 among prehypertensive and hypertensive young adults in Malaysia. Materials and method: In this comparative cross-sectional study, 104 subjects living in Kuantan who gave voluntary consent to participate are recruited and divided into three groups; control (43 subjects), prehypertensive (38 subjects) and mild hypertensive (23 subjects). An optimized droplet digital polymerase chain reaction (ddPCR) was used in the determination of CNV esv27061 in this study. Results: All subjects in the control (n=38; 88.4% gain), prehypertensive (n=33; 86.8% gain) and mild hypertensive (n=21; 91.3% gain) groups had CNV gain (copy number > 2) while 11.6% of control, 13.2% of prehypertensive and 8.7% of mild hypertensive subjects exhibited normal copies (copy number = 2). Conclusion: The present preliminary finding was consistent with the Database of Genomic Variants (DGV) which stated that CNV esv27061 showed more gain than loss.

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Frequency Of Gene Usage and Copy Number Variation Within The Rearranged Immunoglobin Heavy-Chain Variable Locus Based On Immune Repertoire Sequencing

Blood ◽

10.1182/blood.v122.21.3486.3486 ◽

2013 ◽

Vol 122 (21) ◽

pp. 3486-3486

Author(s):

Mark J. Rieder ◽

David Williamson ◽

Anna Sherwood ◽

Ryan O. Emerson ◽

Cindy Desmarais ◽

...

Keyword(s):

Copy Number Variation ◽

Copy Number ◽

Healthy Controls ◽

Immune Repertoire ◽

Employment Equity ◽

Ighv Gene ◽

Gene Usage ◽

Number Variation ◽

Equity Ownership ◽

V Gene

Abstract The human adaptive immune system is composed of both B and T cells that undergo somatic recombination at specific loci to create rearrangements of Variable (V), Diversity (D) and Joining (J) gene segments. For the B-cell immunoglobin receptor heavy-chain (IGH), the CDR3 regions are defined by the VDJ gene segments and nucleotide insertions/deletions at these junctions that create the vast sequence diversity of the IGH repertoire. Characterizing the germline DNA in these regions is impeded by the high sequence similarity between gene segments, mutation and copy-number variation (i.e. large insertions/deletions). Currently, there is a fundamental lack of information about the baseline IGH immune repertoire V gene usage and diversity within healthy human controls. To provide an estimate of this, we sequenced functionally recombined gene segments to infer the underlying gene structure. From a set of 132 healthy controls we sorted C19+/CD27+ B-cells from whole blood and amplified genomic DNA using a highly multiplexed PCR assay that targeted the rearranged IGH receptor locus. Following DNA sequencing and data processing to assign V, D and J gene families and names, we examined the usage frequency of IGHV gene segments across all individuals. We found that of the 98 V gene segments only 56 (57%) were used at a frequency > 0.1%, and ∼10 showed little to no usage (present in<1% of individuals). This data also allowed us to identify two IGHV genes currently annotated as orphons (pseudogenes assigned to an alternate chromosomal location) that had unambiguous functional usage (IGHV4/OR15-8; IGHV3/OR16-09) and therefore must reside at the IGH locus on chromosome 14. Finally, by taking this functional approach we were able to screen all V gene segments for germline copy-number variation (e.g. large insertion/deletion events encompassing individual genes) by looking for an excess of deletion events or modal changes in gene usage. We confirmed that existence of 12 of 15 previously identified deleted IGHV gene segments. Strong deletion evidence was observed for an additional six IGHV genes (IGHV3-NL1, IGHV3-33, IGHV1-24, IGHV4-04, IGHV3-41, IGHV3-35) and ten with highly likely germline deletion events. These data suggest that functional immune profiling of rearranged immune receptors provides a more robust method of identifying individual structural variation and provides insight into the immune repertoire of healthy controls. Disclosures: Rieder: Adaptive Biotechnologies: Employment, Equity Ownership. Williamson:Adaptive Biotechnologies: Employment, Equity Ownership. Sherwood:Adaptive Biotechnologies: Employment, Equity Ownership. Emerson:Adaptive Biotechnologies: Employment, Equity Ownership. Desmarais:Adaptive Biotechnologies: Employment, Equity Ownership. Chung:Adaptive Biotechnologies: Employment, Equity Ownership. Robins:Adaptive Biotechnologies: Consultancy, Equity Ownership, Patents & Royalties. Carlson:Adaptive Biotechnologies: Consultancy, Equity Ownership, Patents & Royalties.

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Prevalence of hypertension among young adults in a Jaipur district of Rajasthan, India

International Journal of Community Medicine and Public Health ◽

10.18203/2394-6040.ijcmph20170266 ◽

2017 ◽

Vol 4 (2) ◽

pp. 424

Author(s):

Uttam Kumar ◽

Om Prakash Sharma ◽

Jaswant Goyal ◽

Saryu Sain ◽

Barkha Gupta ◽

...

Keyword(s):

Blood Pressure ◽

Young Adults ◽

High Blood Pressure ◽

Life Style ◽

Male Students ◽

Cross Sectional Survey ◽

Cross Sectional ◽

History Of ◽

Disease Study ◽

The Impact

Background: Young adults are failed to observe in studies on impact of high blood pressure as they are esteemed to be at a low risk of developing the complication or disease. Study of disease prevalence and their relation with life style habits provide the information required to develop interventional strategies. The objectives were to estimate the prevalence of hypertension among young adults in Jaipur district, Rajasthan and to study the impact of life style habits like tobacco use and alcohol consumption on hypertension.Methods: Data were collected from 390 college students aged 18 years and older in selected degree college by a cross-sectional survey. Blood pressure recordings, anthropometric measurements as well as socio-demographic characteristics were collected.Results: High blood pressure was observed in 152 out of the 390 students (38.97%), of which the 20 (5.13%) are hypertensive, majority were newly diagnosed (65%). Prevalence of high blood pressure among male students was higher (40.29% compared to 37.5% among female students). Out of total 33.85% and 5.12 % of the students were found to pre-hypertensive and hypertensive respectively. Prevalence of hypertension was found higher among those with a history of smoking or alcohol consumption.Conclusions: Majority of students with high blood pressure (hypertensive stage) were previously undiagnosed. A large number of students were in pre-hypertensive stage. Their early identification and right intervention at right time will lessen the impact of high blood pressure in productive age.

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PT349 Droplet digital PCR measurement of copy number variation in genome-wide association regions for blood pressure reveal association with essential hypertension

Global Heart ◽

10.1016/j.gheart.2014.03.2073 ◽

2014 ◽

Vol 9 (1) ◽

pp. e235

Author(s):

Francine Marques ◽

Priscilla R. Prestes ◽

Leonardo B. Pinheiro ◽

Katrina Scurrah ◽

Kerry R. Emslie ◽

...

Keyword(s):

Blood Pressure ◽

Essential Hypertension ◽

Copy Number Variation ◽

Copy Number ◽

Digital Pcr ◽

Droplet Digital Pcr ◽

Genome Wide Association ◽

Genome Wide ◽

Number Variation

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Interaction Effect Between Copy Number Variation in Salivary Amylase Locus (AMY1) and Starch Intake on Glucose Homeostasis in the Malmö Diet and Cancer Cohort

Frontiers in Nutrition ◽

10.3389/fnut.2020.598850 ◽

2021 ◽

Vol 7 ◽

Author(s):

Aida Koder Hamid ◽

Johanna Andersson-Assarsson ◽

Ulrika Ericson ◽

Emily Sonestedt

Keyword(s):

Type 2 Diabetes ◽

Copy Number Variation ◽

Glucose Homeostasis ◽

Copy Number ◽

Inverse Association ◽

Salivary Amylase ◽

Cross Sectional ◽

Copy Numbers ◽

Number Variation

Salivary amylase initiates the digestion of starch and it has been hypothesized that salivary amylase may play a role in the development of insulin resistance and type 2 diabetes. The aim was to examine the interaction between copy number variation in the salivary amylase gene AMY1 and starch intake. We studied 3,624 adults without diabetes or elevated blood glucose in the Malmö Diet Cancer cohort. We assessed the associations and interactions between starch intake, AMY1 copies and glucose homeostasis traits (i.e., fasting plasma glucose, insulin and HOMA-IR) and risk of type 2 diabetes over an average of 18 follow-up years. AMY1 copy number was not associated with glucose, insulin or HOMA-IR. We observed a significant interaction between starch intake and AMY1 copies on insulin and HOMA-IR after adjusting for potential confounders (p < 0.05). The inverse association between starch intake and insulin and HOMA-IR was stronger in the group with 10 or more copies (Ptrend < 0.001). In addition, we observed an inverse association between starch intake and type 2 diabetes in the group with 10 or more copies (ptrend = 0.003), but not in the other groups. This cross-sectional observational study suggests that AMY1 copy numbers might interact with starch intake on glucose homeostasis traits. Interventional studies are required to determine whether individuals with high AMY1 copy numbers may benefit from a high starch intake.

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