Research Article 1

Effects of age and biological age-determining factors on telo- 2

mere lengths of diabetes mellitus type 2 patients. 3

Jawaria A. Tariq1, KaleemUllah Mandokhail2, Naheed Sajjad1, Abrar Hussain5,*, Humera Javaid1, Hummaira Sa- 4
daf1,4, Sadia Javaid1, Abdul Rauf Durrani3 5

1 Department of Biotechnology, Sardar Bahadur Khan Women’s University, Quetta 87300, Pakistan; ja- 6
waria_ali_tariq@yahoo.com (J.A.T.); drnaheedsajjad@gmail.com (N.S.); chokohollikhhf@gmail.com (H.J.); 7
hummairas1@gmail.com (H.S.); rorquel_roller@yahoo.com (S.J.) 8
2 Department of Microbiology, University of Balochistan, Quetta 87300, Pakistan; drkaleemullah@gmail.com 9
(K.M.) 10
3 Provincial Reference Laboratory (PRL), Fatima Jinnah General and Chest Hospital, Quetta, Balochistan; 11
abdulraufdurrani@gmail.com (A.R.D.) 12
4 Silesian University of Technology, and Maria Sklodowska-Curie National Research Institute of Oncology 13
5 Faulty of Life Sciences, , Balochistan University of Information Technology, Engineering and Management 14
Sciences, Quetta 87300, Pakistan; abrar.hussain@buitms.edu.pk (A.H.) 15
* Correspondence: abrar.hussain@buitms.edu.pk (A.H.) 16

Citation: To be added by editorial
staff during production.
Academic Editor: Firstname Last-
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Abstract: Telomere length (TL) undergoes attrition over time indicating the process of ageing and 17
linked to a higher risk of diabetes mellitus type 2 (DM-2). This molecular epidemiological study 18
investigated the correlation between leukocyte TL variations and determinants of molecular ageing 19
in 121 Pakistani DM-2 patients through qPCR assay. The ratio of telomere-repeats to SCG copy 20
number was calculated to estimate the TL in each sample. In this study, smaller mean TL were ob- 21
served in 49.6% of females (6.49±0.89 kb), 3.3% of underweight patients (5.94±0.86 kb), 73% of pa- 22
tients on regular medication (6.51±0.80 kb), 13% with very high stress-levels (6.20±0.93 kb), 31% of 23
smokers (6.26±0.57 kb), 49% of patients with low physical activity (6.47±0.69 kb), 49.6% of hyperten- 24
sive patients (6.21±0.80 kb) and 6.6% of patients with the delayed onset of DM-2 (5.98±1.01 kb). This 25
research indicated no significant correlation (R2=3.872e-4) between TL and age of DM-2 patients. 26
However, a negative correlation (R=-0.164) was observed with the duration of diabetes, with a 27
smaller mean TL (6.10±0.73 kb) in patients having DM-2 for more than 20 years. The lack of correla- 28
tion with age might be influenced by various age-determining factors. This study demonstrated 29
those factors, indicating a weak correlation of TL with the ages of smokers (R 2=0.01), obese patients 30
(R2=0.01); a moderate correlation (R2=0.166) with the ages of hypertensive patients, and a negative 31
correlation with the ages of patients taking medicines regularly (R=-0.05), and high-stress levels (R=- 32
0.087). The study suggests that multiple age-determining factors contribute directly while some 33
have a less-obvious correlation with TL and age of DM-2 patients, challenging TL as the sole marker 34
of ageing; thus, highlighting the need for further research to understand underlying factors and 35
mitigate the effect of ageing or premature ageing in diabetic patients. 36

Received: date Keywords: Telomere Length, T/S Ratio, Diabetes Mellitus type 2, Molecular Ageing, qPCR Assay 37
Revised: date
Accepted: date
Abbreviations: Body Mass Index (BMI); Cycle threshold value (Ct Value); Diabetes Mellitus Type- 38
Published: date
2 (DM-2); Ethylene Diamine Tetra Acetic Acid (EDTA); Kilo Basepair (kb); Quantitative Real-Time 39

Polymerase Chain Reaction (qRT-PCR); Single Copy Gene (SCG); Telomere length (TL). 40
Copyright: © 2023 by the authors. 41
Submitted for possible open access
publication under the terms and 1. Introduction 42
conditions of the Creative Commons
Chronic hyperglycemia is an indication of diabetes mellitus type 2 (DM-2), a hetero- 43
Attribution (CC BY) license
geneous illness manifested by altered insulin production and insulin resistance [1]. The 44
(https://creativecommons.org/license
s/by/4.0/).
DM-2 accounts for >85% of all cases of diabetes mellitus [2]. In addition to the elderly and 45

Molecules 2024, 29, x. https://doi.org/10.3390/xxxxx www.mdpi.com/journal/molecules

Molecules 2024, 29, x FOR PEER REVIEW 2 of 16

middle-aged, young people are becoming more and more affected by this condition, es- 46
pecially in non-Caucasian communities [3]. 47
Genetic susceptibility in different populations and the combination of several envi- 48
ronmental variables cause type 2 diabetes mellitus [4]. Diabetes is one of the major health 49
concerns worldwide especially in Asian countries. Based on recent studies, one in four 50
individuals in the general population in Pakistan has DM-2. Pakistan is a developing na- 51
tion with a high prevalence of diabetes, as shown by a recent survey that found 26% of 52
adults in the general population had the disease [5]. 53
Chromosome ends are shielded against fusion and destruction with the help of DNA- 54
protein complexes called telomeres [6]. Telomere length (or TL) undergoes attrition over 55
time indicating the process (and a potential cause) of ageing in human tissues [7]. Accord- 56
ing to many studies on humans, short TL assessed in leukocytes is linked to a higher risk 57
of age-related conditions such as type 2 diabetes [8] and cardiovascular disease [9], along 58
with the individual’s lifespan and mortality rate [10]. Leukocyte TL is also linked to envi- 59
ronmental exposures (e.g., radiation, smoking cigarettes), health variables (e.g., choles- 60
terol, obesity), and lifestyle factors (e.g., physical activity, Dietary habits) [11]. 61
The accelerated molecular ageing process in diabetic patients may cause by several 62
mechanisms including telomere shortening, accumulation of advanced glycation end 63
products (AGEs), cellular senescence, inflammation, oxidative stress, and epigenetic mod- 64
ifications. These molecular ageing mechanisms can lead to many other age-related disor- 65
ders along with diabetes. 66
Different studies have explained the effect of diabetes on telomere shortening, some 67
suggesting an association between diabetes and accelerated aging at the molecular level 68
[12, 13] while few reported no significant correlation between telomere length and age of 69
DM-2 patients [14]. As no epidemiological data related to Pakistan, associating telomere 70
length with chronological age in diabetes type 2 patients, the scope of this molecular epi- 71
demiological study is to find the telomere length dynamics in leukocytes from diabetic 72
patients and to correlate age-adjusted telomere length variations with various determi- 73
nants of the molecular ageing process in Pakistani DM-2 patients. 74

2. Results 75
In this study, there is no significant correlation (Figure 1) between increasing age and 76
telomere length (because R2=3.872e-4, P-value=0.83) in diabetic patients. 77

78
Figure 1: Correlation between Telomere Length and Age of Diabetic Patients. A weak correlation is 79
presented with a linear regression line. 80
Molecules 2024, 29, x FOR PEER REVIEW 3 of 16

This insignificant correlation is due to many other age-determining factors (Table 1) 81

including obesity (based on BMI), onset of diabetes, smoking habits, physical activity rate, 82
hypertension, stress level, gender, and medication. 83

Table 1: Age-adjusted Telomere Length by Different Age-determining Variables in Type 2 Diabetes 84

Patients of Pakistan 85

Minimum
Telomere
and Maxi- No. of P
Age-Determinants Variables Length %ages R R2
mum Patients value
(kb)
Length (kb)
Male 6.56 ± 0.63 4.95-7.73 60 49.6
Gender 0.621 0.045 0.002
Female 6.49 ± 0.89 4.11-8.05 61 50.4
Underweight 5.94 ± 0.86 4.95-6.90 4 3.3
Normal weight 6.53 ± 0.89 4.11-8.05 37 30.6
Pre-obesity 6.51 ± 0.76 4.64-7.59 47 38.8
Obesity* 0.268 0.102 0.01
Obesity class I 6.62 ± 0.66 5.47-8.01 20 16.5
Obesity class II 6.55 ± 0.60 5.90-7.23 8 6.6
Obesity class III 6.76 ± 0.53 6.39-7.64 5 4.1
Never 7.00 ± 0.61 6.57-7.44 2 1.7
Medication Seldom 6.54 ± 0.74 4.26-7.53 46 38.0 0.586 -0.05 0.002
Regular 6.51 ± 0.80 4.11-8.05 73 60.3
Low 6.54 ± 0.65 4.64-7.64 72 59.5
Moderate 6.95 ± 0.99 5.74-8.05 6 5.0
Stress Level 0.344 -0.087 0.008
High 6.55 ± 0.89 4.11-7.96 30 24.8
Very High 6.20 ± 0.93 4.77-7.44 13 10.7
Primary Smokers 6.26 ± 0.57 4.95-7.44 31 25.6
Secondary Smok-
Smoking Habits 6.72 ± 0.77 4.64-8.01 49 40.5 0.277 0.1 0.01
ers
Non-Smokers 6.50 ± 0.84 4.11-8.05 41 33.9
Low Activity 6.47 ± 0.69 4.64-8.05 49 40.5
Physical Activity Medium Activity 6.59 ± 0.88 4.26-8.01 44 36.4 0.679 0.038 0.001
High Activity 6.53 ± 0.74 4.11-7.73 28 23.1
Hypertensive 6.21 ± 0.80 4.11-7.96 60 49.6
Hypertension 0 0.408 0.166
Non-Hypertensive 6.84 ± 0.60 5.47-8.05 61 50.4
30-39 6.23 ± 1.07 4.26-8.01 17 14.0
40-49 6.60 ± 0.63 4.95-8.05 72 59.5
Disease Onset 50-59 6.70 ± 0.84 4.11-7.73 18 14.9 0.977 0.014 0
60-69 6.44 ± 0.81 5.39-7.59 10 8.3
>70 5.98 ± 1.01 4.64-7.08 4 3.3
<1-4 6.61 ± 0.72 4.77-7.96 60 49.6
5-9 6.57 ± 0.92 4.11-8.05 25 20.7
Duration of Diabe-
10-14 6.46 ±0.76 4.26-7.5 23 19.0 0.072 -0.164 0.027
tes
15-19 6.36 ± 0.73 5.48-7.29 5 4.1
>20 6.10 ± 0.73 4.64-6.89 8 6.6
* According to the WHO standards (2010) 86
** The arithmetic mean (95% CI) was used to calculate the corresponding telomere length in kb, and linear correlation to find the 87
significant value (2-tailed), Pearson correlation coefficient (R), and R square values. 88

A typical data set from leukocytes of 60 males and 61 females in the Pakistani popu- 89
lation with type 2 Diabetes is shown in Figure 2. For the females, the leukocytes had a 90
mean telomere length of 6.56 kb/diploid genome (with the longest telomere length in the 91
age group of 60-64 years old individuals); while in males, leukocytes had a mean TL of 92
Molecules 2024, 29, x FOR PEER REVIEW 4 of 16

6.53 kb/diploid genome (with the longest telomere length is in age group of 50-54 years 93
old individuals). 94
For obesity (Figure 3), the shortest telomere length as well as the longest one were 95
observed in the group of individuals with normal weight, while pre-obese individuals 96
were observed in higher frequency (47 individuals) with a mean telomere length of 6.51 97
kb/diploid genome. 98
Individuals who had never taken medicine for diabetes tend to have longer telomere 99
length as compared to those who seldom take medicines or on a regular basis. However, 100
the individuals with both the smallest and the longest telomere lengths were on regular 101
medication (Figure 4). 102
Diabetic individuals with moderate stress levels tend to have longer telomeres as 103
compared to individuals with higher and very higher stress levels (Figure 5). On average, 104
primary smokers tend to have smaller telomere lengths as compared to secondary smok- 105
ers and non-smokers. However, both the individuals with the smallest and longest telo- 106
mere lengths fall in the group of non-smokers (Figure 6). 107
Individuals with moderate to higher physical activity have longer telomere lengths 108
as compared to individuals with a lower activity rate. On the contrary, the individual with 109
the longest telomere length was not much physically active as compared to the individual 110
with the smallest telomere length (Figure 7). 111
Hypertensive diabetic individuals represented smaller telomere lengths as compared 112
to the non-hypertensive individuals (Figure 8). For the hypertensive individuals, the leu- 113
kocytes had a mean telomere length of 6.21 kb/diploid genome (with the longest telomere 114
length in the age group of 50-54 years old individuals); while in non-hypertensive, leuko- 115
cytes had a mean TL of 6.84 kb/diploid genome (with the longest telomere length is in the 116
age group of 60-64 years old individuals). Moreover, according to this study, the longer 117
the duration of diabetes shorter the telomere length on average (Figure 9 and 10). 118

119
Figure 2: Correlation between gender and age-adjusted telomere length. a) Box-plot showing corre- 120
lation among DM-2 patients and b) Age-adjusted plot for correlation. 121
Molecules 2024, 29, x FOR PEER REVIEW 5 of 16

122

123
Figure 3: Correlation between obesity and age-adjusted telomere length. a) Box-plot showing cor- 124
relation among DM-2 patients and b) Age-adjusted plot for correlation. 125

126
Figure 4: Correlation between medication routine and age-adjusted telomere length. a) Box-plot 127
showing correlation among DM-2 patients and b) Age-adjusted plot for correlation. 128
Molecules 2024, 29, x FOR PEER REVIEW 6 of 16

129
Figure 5: Correlation between stress level and age-adjusted telomere length. a) Box-plot showing 130
correlation among DM-2 patients and b) Age-adjusted plot for correlation. 131

132
Figure 6: Correlation between smoking habits and age-adjusted telomere length. a) Box-plot show- 133
ing correlation among DM-2 patients and b) Age-adjusted plot for correlation. 134
Molecules 2024, 29, x FOR PEER REVIEW 7 of 16

135
Figure 7: Correlation between physical activity levels and age-adjusted telomere length. a) Box-plot 136
showing correlation among DM-2 patients and b) Age-adjusted plot for correlation. 137

138
Figure 8: Correlation between hypertension and age-adjusted telomere length. a) Box-plot showing 139
correlation among DM-2 patients and b) Age-adjusted plot for correlation. 140
Molecules 2024, 29, x FOR PEER REVIEW 8 of 16

141
Figure 9: Correlation between onset of diabetes and age-adjusted telomere length. a) Box-plot show- 142
ing correlation among DM-2 patients and b) Age-adjusted plot for correlation. 143

144
Figure 10: Correlation between duration of diabetes and age-adjusted telomere length. a) Box-plot 145
showing correlation among DM-2 patients and b) Age-adjusted plot for correlation. 146

3. Discussion 147
Telomere length assays help to understand the mechanisms of ageing because telo- 148
mere length serves as a unique cellular and molecular marker for studying the ageing cell. 149
Not only the ageing mechanism, but several studies also supported the theory of associa- 150
tion of telomere length with age-related diseases, cancer, and lifestyle changes (e.g. smok- 151
ing and physical activity). Extensive epidemiological studies on different populations are 152
used to explain or deny the correlations between telomere length and a range of diseases 153
[15]. Where many of diseases are found to be correlated with shorter telomere length in- 154
cluding ischemic heart disease [16], lung cancer among smokers [17], Alzheimer's Dis- 155
ease [18], blood pressure [19], diabetes [20], ageing [21], and dementia [22], many of the 156
studies indicate that few diseases are not significantly correlated with the telomere length 157
Molecules 2024, 29, x FOR PEER REVIEW 9 of 16

including colorectal cancer [23], age-related macular degeneration [16], and death from 158
infections, cardiac or cerebrovascular disease or cancer [24]. 159
Diabetes is a serious, chronic condition affecting the lives of individuals and popula- 160
tions worldwide. In 2017, it was projected to have caused four million deaths worldwide, 161
ranking among the top 10 causes of mortality for people [25]. As of 2019, the global prev- 162
alence of diabetes is estimated to be 9.3% (about 463 million adults aged 20-79 years). 163
China, India, and Pakistan are expected to have the highest number of diabetic individuals 164
in 2045 with a count of 147, 134, and 37 million, respectively [26]. 165
Different researchers have indicated a correlation between shorter leukocyte telo- 166
mere length and diabetes and diabetes-associated complications including impaired glu- 167
cose tolerance and diabetic macroangiopathy [12]. However, it is yet unknown, how dia- 168
betes affects TL in relation to age. While some research [27] have found no age-related 169
telomere length attrition between patients with diabetes mellitus and non-diabetic indi- 170
viduals, others [28] have shown an age effect. An association of shorter telomere length, 171
ageing, and diabetes can be alarming for a country like Pakistan which had a 62% incre- 172
ment [26] in the cases of diabetes (of 20-79 years old individuals) in the past 10 years. 173
However, this research indicated that there is no significant correlation (R2=0.000) between 174
telomere length and age of diabetic patients; but the duration of diabetes tends to have a 175
weak negative correlation (Pearson correlation coefficient R= -0.164) with the telomere 176
length of diabetic individuals indicating that longer the period of disease shorter will be 177
the telomere length (Figure 10). 178
The lack of correlation between telomere length and age of diabetic patients however 179
might be accompanied by several other age-determining factors highlighted in this study. 180
Organ dysfunction brought on by ageing and diabetes is caused by comparable molecular 181
pathways. The abundance of senescent cells in various tissues increases with age, obesity, 182
and diabetes [29]. Although obesity is associated with shorter telomeres overall [30], stud- 183
ies in the elderly found no relation between telomere length and obesity and no relation 184
with mortality [31]. The present study also indicates the negligible correlation (R 2=0.01) 185
between obesity and telomere length variations among diabetic patients. 186
There are studies indicating the association between physical activity levels and te- 187
lomere length dynamics. Cherkas, et al. [32] and Ludlow, et al. [33] independently re- 188
ported a significant relationship between physical activity level and telomere length, such 189
that moderate physical activity levels are correlated with a significantly longer peripheral 190
blood mononuclear cells telomere length in comparison with both the highest and the 191
lowest quartiles of physical activity. The present study also indicates a weak correlation 192
(R2=0.001) between telomere length and physical activity levels of diabetic patients. How- 193
ever, on average individuals with moderate physical activity rate tends to have longer 194
telomere lengths as compared to the individuals with lower or higher activity rates (with 195
few exceptions). 196
Several papers have reported that hypertensive individuals are associated with a 197
shorter telomere length [34]. This study also indicates that hypertensive individuals tend 198
to have shorter telomere lengths as compared to non-hypertensive patients (R2= 0.166 and 199
P< 0.005). 200
Stressful lifestyles caused by hectic work schedules, family issues, financial burdens, 201
or emotional damage have been correlated with telomere length variations. Many studies 202
suggest that telomere attritions caused by stress could lead to premature aging without 203
adequate recovery [35]. The present study also indicates a negative correlation (Pearson 204
correlation coefficient=-0.087, P-value=0.344) between telomere length and stress level of 205
diabetic patients such that individuals with high or very high stress levels have shorter 206
telomere lengths as compared to those having moderate stress levels. 207
Prevention of TL attrition is also associated with healthy life choices, such as not 208
smoking tobacco and drugs or medicines [17, 36]. In this study a weak correlation (R2=0.01 209
and P value=0.277) is observed between telomere length and smoking habits and a nega- 210
Molecules 2024, 29, x FOR PEER REVIEW 10 of 16

tive but weak correlation (Pearson coefficient= -0.05, P value= 0.586) with regular medica- 211
tion of diabetic individuals. Smokers tend to have shorter telomere lengths as compared 212
to secondary smokers or non-smokers and individuals who never had taken medicines 213
for diabetes have longer average telomere lengths (7.0 kb) than those who take medicines 214
seldom (6.54 kb) or regularly (6.51 kb). 215

4. Materials and Methods 216

4.1 Experimental Design 217

The experimental design was planned as suggested by Cawthon [37] and Axelrad, et 218
al. [15] for determining telomere lengths by quantitative PCR. Cell populations from dif- 219
ferent tissues may have different replicative histories and so is the telomere length in those 220
cells. For this research, the telomere length of the leukocytes was measured. For each DNA 221
sample from diabetic patients, the ratio of telomere-repeats copy number (T) to single 222
copy gene copy number (S) was calculated for the estimation of telomere length in the 223
sample. The T/S ratio and telomere length are directly proportional as a primer-DNA 224
binding tendency (during initial PCR cycles) and telomere length are associated directly 225
with each other. Thus, both the copy numbers (T and S) were measured by comparing the 226
difference in cycle threshold (Ct Value), of samples with the primers of telomere and the 227
single copy gene (SCG). 228

4.2 Inclusion Criteria 229

Individuals above the age of 39 years and with a medical history of diabetes mellitus 230
2 were selected for this study. To minimize the effect of extraneous variables (e.g. individ- 231
uals sharing approximately the same environmental conditions, same geographical com- 232
ponents, and no phenotypic expression of any genetic disorder), a prior survey-based 233
analysis was conducted to include individuals sharing most of the common age-related 234
determinants. There are many dietary, demographic, and lifestyle variables and illnesses 235
associated with altered telomere lengths. Therefore, these variables were assessed through 236
a questionnaire-based analysis of the subjects (with their consent). 237

4.3 Sample Collection 238

Fresh blood from 121 Diabetic patients (>39 years of age) was collected voluntarily 239
through standard venipuncture technique in BD Sterile vacutainer blood tubes with 240
EDTA (Becton Dickinson, USA; Cat. No.: 366643). All the samples were processed for 241
DNA extraction protocol on the same day of sample collection to maintain uniformity in 242
the procedure. 243

4.4 DNA Extraction 244

For the optimization of DNA extraction protocol, different methods were scrutinized 245
to assess the integrity and purity of isolated DNA molecules. The organic method by Shen 246
[38] using phenol-chloroform solution was found to extract intact and pure DNA mole- 247
cules showing a sharp bulky band with no smearing during gel electrophoresis. Therefore, 248
all the blood samples were processed through the organic DNA extraction method. Ex- 249
tracted DNA was stored in low Tris-EDTA buffer (TE-4, pH=7.5) at -20 degrees Celsius till 250
further use (not more than 3 days). Before amplification, the quantity and quality of DNA 251
were measured using a NanoDrop spectrophotometer (Thermo Scientific TM, USA; Cat. 252
No.: ND-2000) through the A260/280 ratio. 253
* All the procedures were performed in the biological safety cabinet. 254

4.5 Oligomers 255

All oligomers including standards and primers were diluted in PCR-grade water to 256
a stock concentration of 100pmoles/μl and kept at -20 °C till further required. Working 257
Molecules 2024, 29, x FOR PEER REVIEW 11 of 16

stocks (10pmoles/μl) of all the primers were freshly prepared before starting the reaction 258
and the remaining working primers were stored at 4°C (not longer than 2 weeks). Stand- 259
ards and primers (Supplemental Table 1) for the SCG (β-Globin) and telomeres were used 260
as stated by O'Callaghan and Fenech [39]. 261

4.6 Serial Dilutions 262

Oligomer standards (both telomeric and SCG standards) were serially diluted in 263
PCR-grade water by the dilution factor (1.68) suggested by O'Callaghan and Fenech [39] 264
to generate the standard curve of Ct value for assay as shown in Table 2. 265

Table 2: Serial Dilutions of Standards 266

Telomere Standard (ng/µl) SCG Standard (ng/µl)

6.10 1.8
3.63 1.07
2.16 0.64
1.29 0.38
0.77 0.23
Dilutions of the standard oligomers were then added to the PCR tubes with ultra- 267
clear caps for qPCR assay. Also, 20 ng of plasmid DNA (pBR-322) was added to each tube 268
of serially diluted standards to maintain the overall mass of the DNA molecule. 269

4.7 Normalization 270

Varying DNA concentrations may give different average telomere lengths in each 271
sample. To avoid this problem and also the pipetting error, a DNA normalization was 272
performed by maintaining a consistent concentration of DNA (5ng/ul) in each sample by 273
diluting extracted DNA samples with PCR-grade water as practiced by Axelrad, et al. [15]. 274

4.8 qPCR protocol 275

After diluting all the DNA samples to a final concentration of 5 ng/μl, two master 276
mixes of PCR reagents were prepared: one with telomere forward and reverse primers, 277
and the other with the primer pair of SCG. Aliquot was prepared for no template control 278
[40], standards plus an extra 5% for pipetting error (Supplemental Table 2). 279
All samples were run on a SaCycler-96 (Sacace Biotechnology, Italy) with the SaCy- 280
cler-96 Real-Time PCR V.7.3 (Sacace Biotechnology, Como, Italy). Both Telomere and SCG 281
reactions were run separately using the following program. The reaction program was set 282
as; Initial denaturation of 10 min followed by 35 cycles of 95 degrees Celsius for 15 sec., 60 283
degrees Celsius for 60 sec., and finally a dissociation (or melt) curve. 284

4.9 Data Analysis 285

Amplification was observed in standards and samples according to the procedure 286
previously performed by O'Callaghan and Fenech [39]. The baseline was set (because 287
standards were amplified earlier than the samples) and a standard curve was generated 288
using the Ct readings. 289
Both, telomere and SCG assays were run on each sample. The number of cycles re- 290
quired for the fluorescence detection to attain the exponential curve (Ct Value) varies be- 291
tween these two assays for a single sample due to the varied DNA copy numbers gener- 292
ated in each assay (Figure 11). 293
Molecules 2024, 29, x FOR PEER REVIEW 12 of 16

294
Figure 11: The q PCR Assay. a) Amplification curve of telomeric regions and b) Amplification curve 295
of SCG to evaluate cycle threshold 296

A mean TL of 4,270 bp in leukocytes is equal to one T/S ratio unit (qPCR cycles of the 297
telomere standard run over the cycles of the SCG standard run) [41]. In this study, the 298
ratio (0.98) is equivalent to an average of 4,185 bp. 299

After calculating telomere length for each sample, the effects of different variables 300
(gender, smoking, physical activity, stress level, etc.) on telomere length dynamics in dia- 301
betic patients were assessed by using IBM SPSS Statistics 20. 302

5. Conclusions 303
The RT-qPCR is a powerful tool for telomere assay but detects average telomere 304
length, and not on an individual chromosome basis. This method is also extremely sensi- 305
tive, so cross-contamination was avoided and the method of analysis was planned appro- 306
priately to nullify the chances of erroneous or false results. 307
The study was conducted on DM-2 patients from Pakistan. The statistical measure of 308
this study suggests that there is no significant linear relation between the age of individ- 309
uals with DM-2 and their telomere length. This finding is important because telomere 310
length is often considered a potential marker of the ageing process. The lack of correlation 311
suggests that other factors beyond chronological age may be more influential in determin- 312
ing telomere length, particularly in diabetes mellitus type 2 patients. 313
Through this research, it is concluded that telomere length may not be the only 314
marker for the ageing mechanism but many of the other age-determining factors are 315
worth noting to also contribute to the ageing process; some of which can correlate with 316
telomere length (e.g. gender, hypertension, smoking habit, stress level, onset of diabetes, 317
etc.) directly while some have a non-obvious correlation with telomere length (e.g. obe- 318
sity, duration of diabetes, etc.) of DM-2 patients. So, this study paved the way to determine 319
the underlying factors that can be further studied to lower the effect of ageing or pre- 320
mature ageing in diabetic patients. 321
One more breakthrough of this research includes the further analysis of de-trending 322
cases (individuals with longer telomeres even in older age and individuals with shortest 323
telomere even at younger age) by asking them for their complete medical checkups and 324
through insight through their lifestyle. the person with a shorter telomere length was 325
found to be suffering from invasive stage II breast cancer, while the individual with a 326
longer telomere length never had taken any allopathic medicine in his whole life span. So, 327
if properly organized this assay method (telomere length analysis through qPCR) can also 328
be used as a preliminary diagnostic method for diseases like cancer. 329

6. Recommendations 330
Molecules 2024, 29, x FOR PEER REVIEW 13 of 16

The Real-time qPCR is an efficient method to measure telomere length even for large 331
sample sizes requiring a minimal amount of DNA, so not only to investigate the correla- 332
tion with the age-related disease but must also be used for diagnostic purposes to find the 333
pre-mature ageing in individuals and other diseases like cancer so that such disorders can 334
timely be treated or cured. 335
There is a need for further study to find mechanisms for cellular ageing, senescence, 336
and disease state where this assay method fails to show a significant correlation of telo- 337
mere length with age-related factors and diseases. Such studies will help to shift the focus 338
of future ageing research from disease-oriented studies to cellular- or molecule-oriented 339
studies. 340
Although this study finds a correlation between the onset of diabetes and telomere 341
shortening, leading to the theory that diabetes accelerates the molecular aging process, 342
there is a need for further research to fully understand the underlying mechanisms and 343
the extent of the impact of diabetes on telomere length dynamics. 344
Author Contributions: J.A.T. was involved in the conception, design and validation; J.A.T and A.H. 345
contributed in drafting the study manuscript; K.M. and N.S. were involved in the supervision and 346
co-supervision of the method development, respectively. H.J., H.S., S.J. and A.R.D. provided tech- 347
nical support in sample collection and data handling. All authors have read and agreed to the pub- 348
lished version of the manuscript. 349

Funding: This research was funded by the Higher Education Commission (HEC), Pakistan through 350
the “Prime Minister’s Fee Reimbursement Scheme”. 351

Institutional Review Board Statement: The design, voluntary consent by participants and religious 352
and ethical perspectives of this research were approved by the Departmental Research Committee 353
(of Department of Biotechnology, Sardar Bahadur Khan Women’s University, Pakistan) and Board 354
of Advance Studies and Research (Sardar Bahadur Khan Women’s University, Pakistan). 355

Informed Consent Statement: Informed consent was obtained from all subjects involved in the 356
study. 357
Data Availability Statement: We encourage all authors of articles published in MDPI journals to 358
share their research data. In this section, please provide details regarding where data supporting 359
reported results can be found, including links to publicly archived datasets analyzed or generated 360
during the study. Where no new data were created, or where data is unavailable due to privacy or 361
ethical restrictions, a statement is still required. Suggested Data Availability Statements are availa- 362
ble in section “MDPI Research Data Policies” at https://www.mdpi.com/ethics. 363

Acknowledgments: We acknowledge the volunteers who consented to provide data and blood sam- 364
ples for this research. We are grateful to Dr. Noaman Saeed, CEO, of The Biogene Labs and Diag- 365
nostic, Islamabad for the permission to work in his Lab. 366

Conflicts of Interest: The authors declare no conflicts of interest. The funders had no role in the 367
design of the study; in the collection, analyses, or interpretation of data; in the writing of the manu- 368
script; or in the decision to publish the results. 369

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