3. INTRODUCTION
Hypertension is a significant modifiable risk factor for renal, cardiovascular, and
cerebrovascular disease, as well as the largest underlying cause of worldwide
death and morbidity.
Primary hypertension, also known as essential hypertension, and secondary
hypertension are the two a category into which hypertension is categorized.
Blood pressure is a liberated risk factor for cardiac diseases and progression to
renal failure. Persistently elevated blood pressure levels are associated with
cardiac, vascular, and renal disease.
The pathogenesis of essential hypertension is a complicated and extremely
complex disease. The kidney is both a contributor and a target organ of
hypertension processes, and the disease is a result of the interplay of many organ
systems and numerous independent or interdependent mechanisms.
4. INTRODUCTION
A protein called adducin, which is widely expressed, was first isolated from
human erythrocytes by Gardner and Bennett in 1986 and was then isolated
from bovine brain cells.
It has been shown that ADD1 mutations are linked to hypertension in both
humans and rats.
Numerous human investigations have shown that a mutation in the ADD1 gene
may stimulate sodium and potassium-ATPase (ATPase) activity in renal tubular
cells, increase renal sodium reabsorption, and ultimately result in hypertension
.
5. AIMS AND OBJECTIVES
Objectives of proposed study are given as:
Analyze the role of Alpha Adducin gene Polymorphism in Hypertensive
Patients.
Evaluation of improvement of disease condition in Hypertensive patients by
analyzing single nucleotide polymorphism of ADD1 gene.
To evaluate the Genetic variants of ADD1 gene, how have been implicated as a
risk factor for hypertensive patients
6. METHODOLOGY
Collection of blood samples: Use of Anticoagulant blood vials:
Use of anticoagulant is important for prevention of blood clot formation both in vivo and in vitro.
EDTA can preserve cellular components and blood cells morphology, therefore it has been
recommended historically. Blood can be stored for 12, 24 or 36 hours before processing and can
be stored at -80oC for 20 days and thawed under controlled conditions
Blood test : serum triglycerides and serum cholesterol
DNA extraction: Human genomic DNA was extracted by an inorganic method using NaCl from
3ml of blood samples.
7. DNA QUANTIFICATION
Nano drop is utilized for measurement of DNA or RNA purification. It
functions just like a spectrophotometer. It displays a graph showing the
quantity of UV light absorbed by nucleic acid. First of all, blank the Nano-
drop by using Low T.E. Then, cleaned Nano drop lens carefully by tissue
paper.
Placed about 2 µl of DNA on non-drop lens, following closing of Nano
drop lid and clicking the Measure button. Reading of DNA concentration
was shown on Nano drop along with its O.D (optical density).
8. GENOTYPING AND SINGLE NUCLEOTIDE
POLYMORPHISMS OF ADD1
The Polymerase chain reaction (PCR) was used to detect within at and/or
promoter sites of ADD1 gene single nucleotide polymorphism (SNP).
Primers that intensified a small portion of polymorphism-containing DNA were
used. Single nucleotide polymorphism of ADD1 gene was genotyped by High-
resolution Melt (HRM).
9. SELECTION OF ADD1 SNPS
SNPs of ADD1 gene were selected on the basis of their interaction with disease
susceptibility or prevention. Our research literature focused on the effects of
selected SNPs in hypertensive patients with disease outcomes.
SNP Detection: High-Resolution Melt (HRM):High-Resolution Melt (HRM)
analysis is an operational technique for the identification of single nucleotide
variations in DNA strands by exploring the DNA separation level with DNA
denaturation temperature
10. PRIMER DESIGNING
Primer designing:Form NCBI (National Centre for Biotechnology Information),
sequence of selected single nucleotide polymorphism (under observation) of
ADD1 gene was attained. After initial analysis of human genes, from
bioinformatics tool (www.expasy.ch) primers pairs were designed and are given
in Table.
For primer designing, only exon sequence was used to prevent genomic DNA
contamination. To design primers, sequence from two exons was placed into
another tool i.e. (www.primer3.com). To prevent formation of primer dimer,
primers have least complementarities. GC rich content was about to 50% and
primer length of 20-22 nucleotides. Primer Optimization
11. PRIMER RECONSTITUTION
Reaction Volume (20ul)
PCR mixture
WATER 7ul
Forward primer 1ul
Reverse primer 1ul
PCR master 2X mixture 10ul
DNA 2.0 ul
Total 20ul
12. GEL ELECTROPHORESIS
Agarose gel electrophoresis method was used to visualize and separate the PCR
products on the basis of their size.
It is used for the determination of DNA presence. Fluorescent dye Ethidium bromide is
utilized for this purpose.
After attaching of dye to DNA, it causes fluorescence on gel on the exposure to Ultra
Violet light. Fluorescence level gives the indication about excess or lower concentration
of DNA or RNA.
13. PCR AMPLIFICATION
For ADD1 gene, two specific primers sets were designed to determine SNP genotypes.
Real-time PCR was used to amplify fragment containing SNP by usage of specific
primer pairs. The change from double-stranded to single-stranded DNA is observed by
the fluorescence signal. (Fluorescence dye is emitted and fluorescence signal strength
decreases as temperature increases
14. MUTATION SCANNING: BY REAL-TIME PCR BASED
HIGH RESOLUTION MELTING CURVE ANALYSIS (RT-
HRMCA
HRM is a simple PCR-based technique for identifying DNA sequence variation
by monitoring changes in a DNA duplex's melting.
According to the double-stranded DNA's melting and annealing properties,
several genetic tests have been created.
In real-time PCR, microarray, and fish studies, fluorescent dye-labeled DNA
probes are used to target SNP sequences or mutations.
Due to recent advancements in fluorescent DNA intercalating dye, thermal
cycler temperature control, and data collection methods, melting curve analysis
may now analyze sequence variation with greater precision.
15. HRM PROTOCOL
Real-time PCR was performed on the isolates using a CFX 96 touch
(Bio-Rad, USA) equipment. The reaction took place in a total
volume of 20 l and contained 1 l DNA, 0.5 l forward primer (Link),
0.5 l reverse primer (MSCons-R) (Fermentas, Inqaba Biotech, South
Africa), and 12 l of 2xMaster mixture with SYBR Green dye (Whiz-
Bio, Korea). The total volume was 20 l, and the concentration
ranged from 21 to 93 ng/l. The entire PCR mixture sans the DNA
made up the negative control. Real-time PCR was performed and the
melting curve analysis (Light Cycler®, Roche) was generated with
the following program: Pre-incubation: 96 °C for 2 min,
Amplification: 95 °C for 10 sec, 60 °C for 30 sec, 72 °C for 15 sec.
Melting curves: 99 °C for 0
sec, 70 °C for 15 sec, 99 °C for 0 sec at 0.2 ramp rate and
Cooling: 42 °C for 30 sec.
16. RESULTS AND DISCUSSION
Baseline Characteristics of the Study Population: The baseline and clinical
characteristics of the subjects, hypertensive patients, and healthy controls are
shown in Table 4.1. Older age and sex were significantly associated with
higher risk of hypertension hence to cardiovascular disease risk.
17. TABLE 4.1: BASELINE CHARACTERISTICS OF THE
STUDY POPULATION
Characteristics Normal
n=50
Hypertension
n=50
P-Value
Age, Year 41±6.6 42±9 0.001
Gender
Male 32 (63%) 39 (77) 0.7
Female 20 (38%) 11(23%) 0.3
Laboratory Parameters
BMI, Kg/m2
23.40±1.09 26.4±2.93 0.7
Systolic BP (mmHg) 117.5 ± 0.5 153.4 ± 0.8 0.0001
Diastolic BP (mmHg) 78.2 ± 0.3 97.0 ± 0.5 0.0001
Serum Triglyceride, mg/dl 118.3 ± 2.5 120.8 ± 2.9 0.1
HDL-C, mg/dl 46.5±6.10 39.12±11.1 0.1
LDL-C, mg/dl 78.24±9.12
112.02±42.01
0.1
Serum Cholesterol, mg/dl 146.2±12.10
227.4±54.60
0.1
18. FIGURE: 4.1 COMPARISON OF BP (BLOOD
PRESSURE), TRIGLYCERIDE, CHOLESTEROL
AND BMI BETWEEN NORMALAND
HYPERTENSIVE PATIENTS.
25. REFRENCES
Alam, S., Liyou, N., Davis, D., Tresillian, M., & Johnson, A. G. (2000). The
460Trp polymorphism of the human α-adducin gene is not associated with
isolated systolic
Chait, A. & Subramanian, S. (2000). Hypertension: Pathophysiology, Role of
Genetics, Consequences, and Treatment. Updated 2019, In: Feingold KR,
Anawalt B, Boyce A, et al., editors. South Dartmouth (MA): MDText.com,
Inc.; 2000.
Chandak, G.R., Ward, K.J., Yajnik, C.S. et al. (2006). Triglyceride associated
polymorphisms of the ADD1 gene have very different allele frequencies in
Pune, India compared to Europeans, Journal of BMC Medical Genetics, 7, 76.
https://doi.org/10.1186/1471-2350- 7-76
Chen, H., Ding, S., Zhou, M., Wu, X., Liu, X., Wu, Y., & Liu, D. (2018).
Association of rs662799 in ADD1 with CAD in Chinese Han population,
Journal of BMC cardiovascular disorders, 18 (1),
2.https://doi.org/10.1186/s12872-017-0735-7
