Analysis of the data was based on two sample groups of individuals. These samples included healthy people and hydroxylase deficiency, people. RT-PCR method was used for the project, with the utilization of six primers for the epithelial sodium channel (ENaC) in the human kidney. The data analysis was done to depict the expression of each gene on human. The results are typically normalized to the gene of beta subunit with the exception of the housekeeping gene. The need to use urine samples from the two groups of individuals was to determine the expression of each gene on humans, from the perspective of such samples. To get perfect results, the urine donors had to be free of acute illness and were supposed to be free of any medication during the sampling.
Typically the ENaC has been found to be a heteromeric channel that has three similar subunits that are distinct. These include alpha (α), beta (β), and delta (γ).[i] These three subunits are regulated developmentally. They are also controlled differently by aldosterone. The alpha subunit, in particular, and its variants are expressed within the epithelial cells in the lungs. They are typically suppressed by oxidative stress.
To study the DNA synthesis, for all the subunits, each respective primer had to be studied. This is critical because primers are the basic points for DNA synthesis. The 669-alpha primer consists of both forward and reverses primers on its 12th exon. All alpha primers have 12 exons. The 728-alpha primer has the reverse and forwards primers on is 1st exon. The two primers are shown in figure one and figure 2.
Figure 1: a669- ENaC Primer
Figure 2: a728- ENaC Primer
The mutations of the beta subunit, specifically its last exon, have been found to have the potential of leading to Liddle’s syndrome. Nevertheless, the forward primer process has been noticed between the 4th and 5th exon while the reverse primer process has been noticed in the 5th exon as shown in figure 3 below:
Figure 3: Figure 2: ENaC Beta Primer
In essence, the gain of function mutations in the beta and delta subunits of the epithelial sodium channel has been found to contribute to pseudoaldosteronism, which is also referred to as the Liddle’s syndrome.[ii] Liddle’s syndrome is typically a form of severe salt-sensitive hypertension. It is considered to be a rare and monogenic type of hypertension.
Relatively, the delta primer has more exons than both alpha and beta primers. For the delta subunit, some difference was noticed as well. The forward primer process was noticed between the 5th and 6th exons while the reverse primer process was found to occur in exon 6 as seen in figure 4 below.
Figure 4: Figure 2: ENaC Delta Primer
Other than the various primers, variations are also seen in the respective DNA gels. The alpha-669 ENaC had its DNA gel record a blood pressure of 94bp. This blood pressure is relatively normal. The DNA gel is as shown in figure 5.
Figure 5: Alpha-669 DNA Gel
The alpha-728 DNA gel recorded blood pressure of 150bp. This blood pressure depicts stage 1 of hypertension, which means that the patients’ blood pressure is higher than normal.
Figure 6: Alpha-728 DNA Gel
The cases of delta and gamma DNA Gels saw the subjects’ blood pressure level at 112bp for delta-638/74bp for delta-802 and 136bp respectively. This blood pressure is relatively normal, although it can be associated with medical problems like dizziness especially when subjects are standing. The results are seen in figure 7, figure 8 and figure 9 below.
Figure 7: Delta-638 DNA Gel
Figure 8: Delta-802 DNA Gel
Figure 9: Gamma DNA Gel
The study mainly focused on the beta subunit of the ENaC and the implication of its changes on hypertension.[iii] In this case, the beta-DNA gel was observed to be portraying a normal blood pressure since 77bp was recorded.
Figure 10: DNA gel for Beta ENaC
Generally, the use of DNA gel was to show the potentiality of hypertention with respect to the recorded blood pressure.
To establish the relationship between the beta ENaC subunit in the human kidney and its relation to hypertention, a copy number analysis was used for the DNA copy number variation for the samples. The copy number of a DNA sequence refers to the number of copies of such DNA a genome region. The main aim was to detect the variations, which could cause hypertention. Generally, genetic diseases that have Mendelian transmissions are characterized by some mutations that have rare allelic frequency. A small human population of about 0.1% is usually affected by the associated severe phenotype.[iv] More frequent mutations that are often of single nucleotide polymorphisms (SNPs) are found to have allelic frequencies of 1% or even 2% within the human populations. Other polymorphisms like copy number variations have been considered in this study. From previous studies, it has been found that genetic aspects account for 30% of blood pressure variation in human population. It can thus be asserted that hypertention development depends on the interaction between risk factors and genetic factors. Many genes are found to be associated with the control of blood pressure or essential hypertention.[v]
Regarding the beta gene to which the results were normalized, there are two common beta ENaC variants. These include the G589S and the novel intronic i12-17CT substitution. From the data analysis, it was found that the beta gene was slightly higher among the 21 hydroxylase deficiency group as compared to people within the healthy group based on the copy number values for each case. The β hydroxylase group of people was found to experience a sharp drop in their blood pressure.[vi] The higher value is attributed to the fact that these individuals are already suffering to hypertention. Regarding the same issue, mutation within the DBH gene results to the hydroxylase deficiency. Healthy people do not face the same mutation and thus they are likely to face a smaller effect of the mutation. A general copy number analysis of the β-ENaC showed that most of the subjects were below 50. Only nine subjects have recorded a copy number value above 50. Nevertheless, the two groups seem to depict a close trend in the variations of the beta gene. The results were presented graphically as follows:
Figure 11: Copy number analysis for healthy people
Figure 12: Copy number analysis for 21 hydroxylase deficiency people
Figure 13: Copy number analysis for Beta-ENaC
Further, the amplification of cDNA that is generated from 1ug of human RNA was done for each primer. This was done to measure the expression profile of the beta gene in comparison with other primers. The amplification results for the primers were as shown in figure 4 to figure 11 below.
In the amplification of cDNA generated from 1ug of human RNA, the aim was to show the relationship between the number of PCR cycle and yield in relative fluorescence units (RFU). This test depicted varying patterns of the relationship for every primer between the healthy group and the hydroxylase group of people.
Figure 14: Amplification of cDNA generated from 1 ug of human RNA
In the case of d802-ENaC, it seems that the PCR cycle number does not affect the yield in terms of RFU in the two groups. The effect makes a turn after the 30th cycle. In cycles 1 to 30, zero PCR volume is obtained. The two groups, however, show a different rate of change. The groups, however, appear to show an equal relationship between the PCR cycle number and RFU yield. Nevertheless, only 900 μl were used, which does not provide the chance to monitor beyond the 48th cycle. The two groups have their melt peak at 86 Degrees Celsius. Their RFU yield is however different at their melt peak as shown below
Figure 15: Melt curve analysis of above amplification
For a728- ENaC the trend is different for the two groups. It also turns out that the RFU yield starts at cycle 30. The rate is different for the two groups, but become the same at cycle 36. After cycle 38, the difference between the two groups is infinitely increasing as shown below.
Figure 16: Amplification of cDNA generated from 1 ug of human RNA
Figure 17: Melt curve analysis of the above amplification
Figure 18: Amplification of cDNA generated from 1 mg of human RNA for the beta gene
From figure 8 above, the melt curves (for healthy and hydroxylase deficiency groups) are leveled at zero for the RFU value until the 27th cycle. The two groups then depict a change in RFU at different rates. At RFU of 650 and at the 38th cycle, the two groups are showing a similar rate. The two groups did not seem to attain a maximum RFU even at the 50th cycle. Nevertheless, a melt curve analysis of the above amplification depicted different results as shown in figure 5 below.
Figure 19: Melt Curve analysis of the above amplification.
Figure 5 shows a melt curve for the two groups, but of the above amplification. The maximum RFU is reached at a temperature of 80o Celsius. The RFU then drops at a high rate until it levels at zero RFU after a temperature of 83 o Celsius. The amplification of cDNA was mainly done to obtain the length of RNA transcripts within the cells.[vii] The production of a cDNA copy of the required RNA sequence was successfully achieved.
The amplification of cDNA is a bit different. The RFU yield starts at cycle 24. This is far much lower than in both beta and alpha subunits. The difference in the curves keeps on widening as the yield volume and cycle number increase. This happens as depicted in figure 10 below.
Figure 20: Amplification of cDNA generated from 1 ug of human RNA for g- ENaC
Figure 21: Melt Curve analysis of above amplification.
Figure 11 shows a melt curve for the two groups, but of the above amplification. The maximum RFU is reached at a temperature of 82.5o Celsius. The RFU then drops at a high rate until it levels at zero RFU after a temperature of about 85.5 o Celsius. The amplification of cDNA was mainly done to obtain the length of RNA transcripts within the cells.[viii] The production of a cDNA copy of the required RNA sequence was successfully achieved.
Further, based on the data analysis, it can be established that the beta channel is a non-voltage and sensitive ion channel that is permeable and inhibited by the diuretic amiloride. The beta channel mediates the electrodiffusion of luminal sodium, including water that follows osmotically. This happens through the epithelial cells’ apical membrane. The channel is found to play a critical role in the pressure homeostasis of both blood and electrolyte. The beta channel also provides immense help in the liquid homeostasis of the airway surface. This aspect is important because it offers a proper mucus clearance. The channel also helps in controlling the re-absorption of Na in the human kidney, the colon, the lungs, as well as in the sweat glands. Finally, it plays a major role in the perception of taste.[ix]
Generally, the channel is end-effector within the rennin-angiotensin-aldosterone system. It resides within the apical plasma membrane. The apical plasma membrane is found in the renal cortical collecting duct. Re-absorption of sodium ions through the ENaC happens in collecting ducts of the renal cortical. The re-absorption is typically the final step for the sodium ion balance regarding renal adjustment. The ENaC is found to be playing an important role regarding the modulation of Na+ homeostasis thereby attributing the function to chronic blood pressure.[x]
From the figures depicting the results of data analysis, it is clear that the two groups did not show sharp differences in their expression of the ENaC beta subunit. While the group of people with the disease was used as a control, their results from their samples made it clear that hypertension was not solely related to the ENaC beta subunit. The rate at which the variations of the beta gene occurred among the two groups was almost similar. It can thus be asserted that hypertension is caused by a number of factors, besides the effects of the beta gene of the ENaC.[xi] Besides, it was previously mentioned that hypertension comes as a result of many factors. The factors are both hereditary and risk-related. The aspect of heredity reflects the effects occurring from the mutation of the respective genes. Since the beta gene seems to be equally occurring in people within the two groups, it is difficult to relate it to hypertension without considering other genes and risk factors as well.
[i] Basson, Jacob, Jeannette Simon, and D. C. Rao. “Between Candidate Genes and Whole Genomes: Time for Alternative Approaches in Blood Pressure Genetics.” Curr Hypertens Rep, vol.14, 2012: 46–61.
[ii] Tamura, H, et al. “Liddle disease caused by a missense mutation of the beta subunit of the epithelial sodium channel gene.” J Clin Invest, Vol. 97, 1996: 1780-1784.
[iii] Kawahara, Katsumasa, and Kouju Kamata. “The kidney and hypertension.” Clin Exp Nephrol, vol.16, 2012: 3–7.
[iv] Kawahara, Katsumasa, and Kouju Kamata. “The kidney and hypertension.” Clin Exp Nephrol, vol.16, 2012: 3–7.
[v] Kawahara, Katsumasa, and Kouju Kamata. “The kidney and hypertension.” Clin Exp Nephrol, vol.16, 2012: 3–7.
[vi] Persu, Alexandre, et al. “Genetic Analysis of the beta Subunit of the Epithelial Na+ Channel in Essential Hypertension.” Hypertension, vol.32 No.1, 1998: 129-137.
[vii] Schild, L, E Schneeberger, and I. Gautschi. “Identification of amino acid residues in the alpha, beta and gamma subunits of the epithelial sodium channel (ENaC) involved in amiloride block and ion permeation.” J Gen Physiol, Vol.109, 1997: 15-26.
[viii] Schild, L, E Schneeberger, and I. Gautschi. “Identification of amino acid residues in the alpha, beta and gamma subunits of the epithelial sodium channel (ENaC) involved in amiloride block and ion permeation.” J Gen Physiol, Vol.109, 1997: 15-26.
[ix] Hansson, JH, et al. “Hypertension caused by a truncated epithelial sodium channel gamma subunit; genetic heterogeneity of Liddle syndrome.” Nat Genet, Vol.11, 1995: 76.
[x] Hummler, Edith. “Epithelial Sodium Channel, Salt Intake, and Hypertension.” Current Hypertension Reports, Vol. 5, 2003: 11–18.
[xi] Dong, YB, HD Zhu, and EH Baker. “T594M and G442V polymorphisms of the sodium channel beta subunit and hypertension in a black population.” J Hum Hypertens, Vol.15, 2001: 425–430.
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