3.1 Demographic Information of the Study Population
A total hundred and twenty (120) HbE/β thalassemia patients were recruited with complete information including hematological data, age of first transfusion, transfusion interval, history of splenomegaly or splenectomy, etc. Demographic data of the study participants are shown in Table 3.
Parameters
|
Total Number of Patients(n=120)
|
Age
|
Range
|
3 years to 65 years
|
Mean ±SD
|
19.66±10.22
|
Male
|
Numbers(Percentage)
|
68(56.6%)
|
Mean ±SD
|
20.85±11.27
|
Female
|
Numbers(Percentage)
|
52(43.3%)
|
Mean ±SD
|
18.13±8.53
|
Table 3: Demographic data of the study
3.2 Severity of the disease among three groups of HbE/β-Thalassemia patients
The age of first blood transfusion (months) and transfusion interval (Days) of the three groups are summarized in Table 4. The NTD/mild group has the highest mean±SD of transfusion interval as well as the first age of transfusion. On the other hand, the Severe group has the lowest first age of transfusion (months) and transfusion interval (Days).
Types of Group
|
First Age of Transfusion (Months)
Mean±SD
|
Transfusion Interval (Days)
Mean±SD
|
Group-1: NTD/Mild
|
141.79±131.5
|
100.88±91.48
|
Group-2: Moderate
|
33.76±44.3
|
36.14±41.29
|
Group-3: Severe
|
17.16±16.11
|
22.23±9.79
|
Table 4: Severity of the disease among the HbE/β-Thalassemia patients enrolled in the Study.
3.3 Comparisons of Hematological parameters among three groups
There was no significant difference in HGB and RDW levels. When compared among all three groups, there were statistically significant differences in MCV and MCH levels (P <0.0001, Table-5 & Fig-1).
Group
|
HGB(g/dl)
|
RDW(%)
|
MCV(fl/red cell)
|
MCH(pg/red cell)
|
Hemoglobin-A
|
Hemoglobin-A2
|
Hemoglobin-F
|
Hemoglobin-E
|
NTD
|
7.88±1.23
|
27.05±8.5
|
64.11±7.24
|
20.21±2.9
|
2.27±2.81
|
0.33±0.1
|
2.03±2.05
|
2.88±1.47
|
Moderate
|
7.69±1.44
|
24.24±8.87
|
69.46±6.85
|
21.99±3.08
|
5.33±2.61
|
0.27±0.1
|
0.58±0.78
|
1.36±1.1
|
Severe
|
6.82±1.03
|
22.97±7.59
|
73.26±3.82
|
23.28±2.27
|
4.39±2.42
|
0.23±0.07
|
0.84±1.27
|
1.35±0.97
|
P Value
|
0.009
|
0.0439
|
<0.0001
|
<0.0001
|
<0.0001
|
0.0001
|
<0.0001
|
1.35±0.97
|
Table 5: Comparisons of Hematological parameters among three groups
The hemoglobin variants such as HbA, HbA2, HbF, and HbE were significantly different (p-value- <0.0001, 0.0001, <0.0001, and <0.0001 respectively) among Mild, moderate, and Severe patients. Table 5 shows that HbA2, HbF, and HbE levels were highest in NTD/mild groups, while the lowest HbA2 and HbE levels were in severe groups, but HbF was higher than in moderate groups. On the other hand, unexpectedly moderate groups and severe groups show the highest HbA level.
3.4 Distribution of allele and genotype frequencies of two SNPs
3.4.1 Allele Distributions among HbE/β-Thalassemia patients
The genotype results of two SNPs using the PCR-HRM method were 100% consistent with direct sequencing (Fig. 2). SNP rs28384513 of HBS1L-MYB was detected by Sanger Sequencing. In this study, Major allele ‘T’ and minor allele ‘G’ were detected in all three statuses i.e. homozygous ‘TT’, Heterozygous ‘TG’, and Homozygous ‘GG’ in the study population (Fig. 1). In the case of SNP rs4895441 of HBS1L-MYB, heterozygous AG, and homozygous major allele ‘AA’ were found but no homozygous allele ‘GG’ had been found (Fig. 2). Secondly, the sequenced samples were used as reference samples with known genotypes, and the rest of the samples were tested for the presence of the two SNPs (rs28384513 & rs4895441) by Real-time PCR followed by High-Resolution Melting (HRM) Curve Analysis in the patients of HbE/β-Thalassemia. These temperature-shifted curves showed the differences in melting temperatures in the presence of the polymorphic alleles. Blue curves in Figure-3.9 showed homozygous ‘TT’ alleles, Green showed homozygous ‘GG’ alleles and Red showed heterozygous ‘TG’ alleles of rs28384513 SNP of HBS1L-MYB gene in HbE/β-thalassemia patients (Fig 2).
In our population, in rs28384513, homozygous ‘TT’ and ‘GG’ and heterozygous ‘TG’ had been found which comprised 30% of ‘TT’ alleles and 70% of ‘TG+GG’ alleles. On the other hand in rs-4895441, about 79% AA alleles and 21% AG alleles had been reported in this study among the enrolled patients Figure-3.
3.4.2 Allele frequency among HbE/β-Thalassemia patients
Among one hundred and twenty (120) HbE/β-Thalassemia patients, in the case of rs2838513 NTD/mild patients carried the highest TG+GG alleles compared to the other two groups. On the other hand in the case of rs4895441 Homozygous major ‘AA’ showed a greater percentage compared to the heterozygous ‘AG’ allele. That means rs28384513 (T>G) is more frequent in our population (Table 6).
Locus
|
Genotype
|
NTD/Mild
|
Moderate
|
Severe
|
rs-28384513(T>G)
|
TT
|
13 (10.83%)
|
19 (15.83%)
|
5 (4.17%)
|
TG+GG
|
31 (25.83%)
|
30 (25%)
|
22 (18.33%)
|
rs-4895441(A>G)
|
AA
|
32 (26.67%)
|
40 (33.33%)
|
22(18.33%)
|
AG
|
12 (10%)
|
9(7.5%)
|
5 (4.17%)
|
Table 6: Allele Frequency of rs-28384513 & rs-4895441 among three groups.
3.5 Association of SNPs in HBS1L-MYB with Disease Severity of HbE/β-Thalassemia patients
3.5.1 Comparison of fetal hemoglobin level between HbE/β-Thalassemia patient groups with different SNPs of HBS1L-MYB
In the case of rs-28384513, ‘TG+GG’ alleles showed the highest HbF values as expected than ‘TT’ alleles while significant differences had been found in HbF (P value 0.01). Similarly, in rs-4895441 both ‘AA’ and ‘AG’ alleles showed expected statistically significant differences of HbF (P= 0.03), shown in (Table 7). The comparison of Mean ± SD of HbF(g/dl) was done by T-test and the results were shown in figure 4.
Gene(Choromosoe)
|
Locus
|
Genotype
|
Numbers
|
Mean±SD(HbF)
|
t test (p)
|
HBS1L-MYB(6q23)
|
rs-28384513 (T>G)
|
TT
|
37(31%)
|
0.87±1.1
|
0.01
|
TG+GG
|
83(69%)
|
1.29±1.63
|
HBS1L-MYB(6q23)
|
rs-4895441 (A>G)
|
AA
|
95(79%)
|
1.19±1.65
|
0.03
|
AG
|
25(21%)
|
1.49±1.7
|
Table 7: Comparison of fetal hemoglobin level among HbE/β-Thalassemia patients with different SNPs of HBS1L-MYB.
3.5.2 Correlation Studies of HBF Concentration and Disease Severity
There is a significant correlation of HBF (g/dl) with transfusion interval (Days) including r = 0.38, R squared = 0.14, and P <0.0001*, and a negatively significant correlation was found with Clinical Score (r = -0.31, R squared = 0.0.09 and P value 0.0003*).
However, less correlation was found between HBF (g/dl) and age of first blood transfusion where r = 0.26, R squared = 0.07, and P = 0.0028*. So it can be said that there was a significant but less correlation found of HbF (g/dl) with the age of first blood transfusion (Figure-3.14).
3.6 Demographic, Hematological information and Allele Frequency among healthy Individuals.
Forty (40) healthy controls including 22(55%) males and 18(45%) females were with a mean age of 21.64±3.31 years recruited in this study. All of them had completely normal hematological and hemoglobin parameters. In SNP(rs-28384513), 31% homozygous major allele TT, 46% heterozygous minor allele TG, and 23% homozygous minor allele GG were found. In SNP (rs-4895441), 75% homozygous major allele AA and 25% heterozygous minor allele AG were found (Figure 6).
3.7 Frequency Distribution of rs-28384513 & rs-4895441 SNP of HBS1L-MYB in total study population.
The minor allele frequency of rs28384513 ‘G’ and rs4895441 ‘G’ in the HBS1L-MYB was found as 0.43 and 0.11 respectively in our study population.
Genotype
|
Total Sample(160)
|
Allele Frequency of rs28384513
|
T allele
|
184(0.58)
|
G allele
|
136(0.43)
|
Allele Frequency of rs28384513
|
A allele
|
285(0.89)
|
G allele
|
35(0.11)
|
Table 8: Allele frequency of total study population