Association of Leukocyte Telomere Length and the Risk of Disease Severity and Metabolic Comorbidities in Arab Patients with Psoriasis

Psoriasis is an immune-mediated inflammatory disease of the skin. The main feature of psoriasis is chronic inflammation, which causes sustained skin inflammation leading to defects in keratinocyte proliferation and differentiation. Histological examination of the psoriatic plaque indicated epidermal hyperplasia, which was overwhelmed by inflammatory infiltrates containing dermal dendritic cells and immune cells [1]. However, inflammation is not localized in psoriatic skin but can affect different organ systems. Therefore, patients with psoriasis exhibit increased metabolic comorbidities, including cardiovascular disease (CVD), hyperlipidemia, hypertension, type 2 diabetes (T2D), and obesity [2]. Several studies have reported a higher prevalence of metabolic comorbidities with psoriasis severity [1], as assessed using the Psoriasis Area Severity Index (PASI).

Several studies have associated telomere shortening with various diseases, such as CVD [3, 4], obesity [5], T2D [6], cancer [7], and metabolic syndrome [8], suggesting that telomere length can be used as a biomarker. Telomeres are ribonucleoprotein structures located at the ends of eukaryotic chromosomes and play a critical role in maintaining the structural integrity of chromosomes. It is composed of long hexameric (TTAGGG)ⁿ DNA repeats that shorten with cell division [9]. Following multiple rounds of division, cells with critically short telomeres enter replicative senescence [10]. Telomeric regions contain a high guanine content; therefore, they are more susceptible to oxidative damage. In this region, the level of oxidized purine is seven times higher than that in genomic DNA [11]. The 8-oxodG molecule binds inadequately to adenine, creating transversions of GC-TA that create single-strand breaks, subsequently causing inadequate replication of telomeric DNA and enhancement of telomere shortening [11, 12]. Therefore, telomere length is clinically important for human health and is considered a biomarker of cellular aging. Recently, telomere attrition has been implicated in inflammatory diseases, such as psoriasis [13], atopic dermatitis [13], inflammatory bowel disease [14], and rheumatoid arthritis [15].

Chakravarti et al. [16] showed that mice engineered to experience telomere dysfunction in the intestinal epithelium presented an inflammatory bowel disease-like condition with upregulation of the precursor of interleukin 18 (pro-IL-18), which is the main enhancer of inflammatory bowel disease [16]. Therefore, telomere dysfunction can drive inflammatory processes.

To date, few studies have examined leukocyte telomere length (LTL) in inflammatory diseases, including psoriasis. In relation to psoriasis, a pilot study consisting of 200 patients with different inflammatory diseases, including psoriasis, psoriasis arthritis, osteoarthritis, and ankylosing spondylitis, concluded that LTL in the psoriasis group was significantly shorter than that in healthy controls and other groups [17]. Another study showed that telomere length in peripheral blood mononuclear cells and purified T cells from 16 psoriasis patients was significantly reduced [13]. In contrast, Beranek et al. [18] reported that the telomere length per chromosome was significantly higher in the whole-cell DNA of psoriasis patients than in controls, and the oxidative damage of nucleic acids was higher in the patient’s serum [18].

In this study, LTL was associated with the risk of disease severity and metabolic comorbidities in Arab patients with psoriasis. The content of 8-oxoGua, of telomere, which is the main oxidative damage biomarker, was also measured.

This case-control study included 68 patients with psoriasis and 42 normal controls (NCs). The NC group consisted of 42 healthy blood donors (20 males and 22 females; age range 24–69 years; mean age, 40 years ± 1.6). NCs were recruited from the Central Blood Bank, State of Kuwait, and they were free of inflammatory and systemic diseases.

The psoriasis group consisted of 68 patients (41 males and 27 females; age range 18–76 years; mean, 39 years ± 1.6). Patients with psoriasis were recruited from the dermatology outpatient clinic at Suaid Al-Sabah Medical Center, State of Kuwait.

A dermatologist assessed the severity of psoriasis in each patient using the PASI (score range, 0–72) [19]. Psoriasis scores of up to 10 are considered a mild index, a score of 10–20 is considered a moderate-to-severe index, and a score above 20 is considered a severe index. A questionnaire was administered to each patient, which included age and metabolic comorbidities including T2D, hypertension, dyslipidemia, and obesity. Body mass index (BMI) was determined according to the BMI formula: weight (kg)/height² (m²) and classified as underweight (BMI <18.5 kg/m²), normal (18.5 > BMI <24.9 kg/m²), overweight (29.9 > BMI >25 kg/m²), and obese (BMI >30 kg/m²) [20]. Clinical and biochemical data were obtained from the medical records of patients and controls. The presence of metabolic disorders was based on the definition provided by the International Diabetes Foundation in 2004 [21].

This study complied with the Declaration of Helsinki guidelines. This study was approved by the Ethics Committees of Kuwait University and the Ministry of Health (State of Kuwait) (2016/496). All the participants provided written informed consent.

Blood samples (5 mL) were collected from each participant using ethylenediamine tetra-acetic acid (EDTA). Blood was immediately centrifuged at 3,000 rpm at 4°C for 10 min to isolate the buffy coat containing leukocytes. Genomic DNA was isolated from leukocytes using the QIAamp DNA Mini Kit (QIAGEN, Germany) according to the manufacturer’s instructions. Extracted DNA was quantified using a NanoDrop 100 system (Thermo Fisher Scientific).

LTL was quantified using qPCR as described by Cawthon [22]. Briefly, the relative telomere length was determined as the ratio of telomere copy number (T) to single gene copy number (S) for each sample. Five DNA samples from controls were evenly pooled and used as reference DNA that was serially diluted (from 8 to 0.25 ng/μL) for the standard curve. The primers for the telomeres were Tel-forward (5′ GGT TTT TGA GGG TGA GGG TGA GGG TGA GGG TGA GGG T 3′) and Tel-reverse (5′ TCC CGA CTA TCC CTA TCC CTA TCC CTA TCC CTA TCC CTA 3′). The primers for the single-copy gene were β2M forward (5′ CCAGCA GAG AAT GGA AAG TCA A3′) and β2M reverse (5′ TCT CTCTCC ATT CTT CAG TAA GTC AAC T3′). Each well contained 20 μL PCR reactions with 20 ng of DNA, SYBR Green master mix (Applied Biosystems), and 500 nm of specific primers. The assay was performed in a 96-well plate using a 9700HT Fast Real-Time PCR System (Applied Biosystems). PCR amplification was performed using the following program: one cycle at 95°C for 10 min, followed by 35 cycles at 95°C for 30 s, 58°C for 1 min, and 72°C for 10 s. Melting point curve analysis was performed using one cycle at 95°C for 5 s, 70°C for 1 min and one cycle at 40°C for 30 s to determine the specificity of all reactions. All samples were measured in triplicate, the mean of the results was analyzed statistically, and the negative control (sample without DNA) was included in each run. The ratio of telomere content (T) to β2M content (S) was calculated as T/S = 2^−∆Ct (∆Ct = average Ct_telomere − average Ct_β2M). The median telomere length was used as the cutoff for the long and short telomere length subgroups in the psoriasis group.

Guanine is most susceptible to oxidative damage, making the oxidation products of guanine the main biomarkers for the measurement of free radical-induced DNA damage. Formamidopyrimidine-DNA glycosylase (FPG) is an enzyme that recognizes specifically oxidized purines, including 8-oxoGua. FPG cleaves DNA lesions, causing a reduction in amplification by Taq polymerase.

Oxidative damage to DNA was quantified using qPCR [23, 24]. DNA lesions were measured after digestion with the FPG enzyme and evaluated as ∆Ct (∆Ct = Ct _treated − Ct _untreated). The increased quantity of oxidized purine nucleotides reflects the level of oxidative damage in the telomeres. A higher ∆Ct value indicates higher levels of oxidative damage.

The FPG reaction includes 100 ng of DNA sample digested with 1 unit of FPG in 1xNEB buffer at 37°C for 1 h. The treated DNA was then amplified by qPCR under the same conditions mentioned in the previous section.

A two-tailed unpaired t test was used to evaluate significant differences in LTL between groups. χ² test was used to determine the statistical differences between categorical variables. Logistic regression was performed to evaluate the odds ratios (ORs) and 95% confidence intervals (CIs) and was adjusted for age and sex. Statistical analysis was performed using SPSS software version 25 (IBM SPSS Statistics, USA). Statistical significance was defined as a p value <0.05.

In total, 110 individuals were enrolled in the study. The participant characteristics are presented in Table 1. There was no statistically significant difference between the patients with psoriasis and NC groups in terms of mean age (39 ± 1.6, and 40 ± 1.6, respectively) and sex distribution (41 males, 27 females; 20 males, 22 females respectively). The mean psoriasis severity was 20 ± 1.4. According to the severity index (PASI), 27 patients with psoriasis had mild scores with a mean of 5.9 ± 0.5, and 41 had moderate-to-severe scores with a mean of 25.5 ± 1.6. Thirty patients with psoriasis had metabolic comorbidity including T2D, hypertension, dyslipidemia, and obesity (Table 1). The average mean BMI of patients with psoriasis (83.8 ± 2.5) was significantly higher than that of NCs (25 ± 2, p < 0.001). The fasting blood glucose level of patients with psoriasis (7.4 ± 0.7) was also significantly higher than that of NCs (5.2 ± 0.09, p = 0.008). The lipid profile of patients with psoriasis including cholesterol (3 ± 0.1 mmol/L), HDL (0.9 ± 0.06 mmol/L), triglyceride (1.9 ± 0.1 mmol/L) was significantly different than lipid profile of NCs including cholesterol (4.2 ± 0.2 mmol/L, p = 0.01), HDL (1.3 ± 0.01 mmol/L, p = 0.02), and triglyceride (0.8 ± 0.05 mmol/L, p < 0.001). Systolic blood pressure was significantly higher in patients with psoriasis (146 ± 5 mm Hg) than in NCs (114 ± 4 mm Hg, p < 0.001).

Overall, LTL was significantly shorter in patients with psoriasis (0.92 ± 0.05) than in NCs (1.32 ± 0.2, p = 0.03) (Table 1; Fig. 1). The psoriasis group was subdivided according to the severity index (mild and moderate-to-severe) and metabolic comorbidities (with and without). The association between LTL among the groups was analyzed. The results showed a significantly shorter LTL in patients with a moderate-to-severe index (0.9 ± 0.3) compared to those with a mild index (1.2 ± 0.5, p = 0.03, Fig. 2). The same result was observed in a group of patients with metabolic comorbidities (0.77 ± 0.07) compared to those without (1.05 ± 0.05, p = 0.003, Fig. 3).

LTL was dichotomized with the median value in the controls into short and long telomere length groups (median = 0.9) [25]. A logistic regression model was used to calculate the relationship between LTL and risk of psoriasis severity and metabolic comorbidities (Table 2). Stratification analyses of the association between LTL and patients with moderate-to-severe index (OR = 6.98, 95% CI = 2.3–20.8; p = 0.001) showed a significant elevation of the risk with a short LTL (≤0.9). Patients with metabolic comorbidities showed also higher risk of short LTL (OR = 2.89; 95% CI = 1.02–8.2, p = 0.04).

The content of 8-oxoGua in telomeres was determined using qPCR. The results showed that the mean value of oxidative damage in patients with psoriasis (5.7 ± 1.6) was significantly higher than that in NCs (1.2 ± 0.4, p = 0.015) (Table 1). There was no significant correlation between LTL and the content of oxidative damage (OR = −0.29, 95% CI = −0.71 to 0.28, p = 0.32).

In this study, LTL was investigated in 68 patients with psoriasis and in 42 NCs. Overall, LTL was significantly shorter in patients with psoriasis than in the NCs (p = 0.032). Stratification of patients according to severity index and metabolic comorbidities showed that LTL was significantly shorter in patients with moderate-to-severe index than in those with mild index (p = 0.03) and in patients with metabolic comorbidities than in those without (p = 0.003). Using the median LTL (0.9) of the controls as the cutoff value, patients with short LTL (≤0.9) were associated with a trend of moderate-to-severe conditions (OR = 6.98, 95% CI = 2.3–20.8; p = 0.001) and a higher risk of metabolic comorbidities (OR = 2.89; 95% CI = 1.02–8.2, p = 0.04).

In addition, the results showed that the content of oxidative damage of telomeres was significantly higher in the patients with psoriasis than in the NCs (p = 0.015), and there was no significant correlation between LTL and the content of oxidative damage (OR = −0.29, 95% CI = −0.71 to 0.28, p = 0.32).

LTL has been found to be a useful biomarker for assessing individual susceptibility to various diseases [3, 26‒28]. For instance, telomere shortening has been reported to be an independent predictor of future coronary heart disease events in middle-aged men [6] and affects the cellular metabolic rate, which causes the progression of metabolic syndrome [3]. Meta-analysis studies based on human populations involving different types of cancer have concluded that shorter LTL is significantly associated with an increased risk of cancer and mortality [29].

Few studies have demonstrated an association between LTL and psoriasis. However, to our knowledge, no previous study has examined the association between LTL and the risk of disease severity and metabolic comorbidities in Arab patients with psoriasis. Moreover, the content of oxidative damage to telomeres was significantly higher in patients with psoriasis than in the NCs.

In line with our results, a few studies have reported shortening of LTL in patients with psoriasis. Wu et al. [13] concluded that the telomere length of peripheral blood mononuclear cell and T-cell subsets was significantly reduced in the blood of 16 patients with psoriasis compared with that in NCs. Moreover, a pilot study reported a significantly shorter LTL in patients with psoriasis than in healthy controls and the spondyloarthritis group [17]. Conversely, Beranek et al. [18] reported longer telomeres per chromosome in psoriatic patients than in NCs. The differences between the two studies could be attributed to the different techniques used.

In this study, we reported that short LTL (≤0.9) was associated with a higher risk of severe conditions and a higher risk of metabolic comorbidities. In contrast, Coussens et al. [30] did not support LTL as a biomarker to predict metabolic syndrome or CVD in patients with psoriasis; however, this study has several limitations, including a small population size, lack of healthy controls, and low number of patients with metabolic syndrome.

Additionally, this study revealed that oxidative damage to telomeres is significantly elevated in patients with psoriasis compared to that in NCs. This outcome agrees with that of Beranek et al. [18], who reported that the oxidative damage of nucleic acids was higher in patients’ serum than in controls. This study failed to report a significant correlation between LTL and oxidative damage due to the small sample size included in this study.

Biochemical studies have shown that TTAGGG repeats of telomeres are preferred sites for oxidative damage because of the high number of G-C repetitions that are highly sensitive to oxidative stress [11, 31], which can affect the structure of telomere proteins, such as TRF1, TRF2, and POT1. Fifty percentage of telomeric protein levels were reduced by a single 8-oxodG lesion, shortening the telomere length and leading to telomeric instability [32].

Therefore, telomere dysfunction is commonly associated with inflammatory diseases such as liver cirrhosis [33], inflammatory bowel disease [14], and kidney fibrosis [34] which may increase the risk of developing cancer in patients. Chakravarti et al. [16] studied mice with telomere dysfunction in intestinal cells. The authors concluded that these mice had shorter telomeres and increased intestinal inflammation. Gastrointestinal inflammation has also been observed in samples from pediatric patients with telomere defects [35]. Previous studies suggested that inflammation is driven by telomere instability. In fact, telomere alteration activates the ATM protein. This, in turn, leads to stimulation of a protein called YAP1, which controls the expression of pro-IL-18. Caspase-1 processes pro-IL-18 into IL-18, which stimulates inflammation and recruits immune cells [16]. Increased IL-18 expression in keratinocytes has been reported in psoriatic lesions and is correlated with disease duration and severity [36].

In conclusion, this study concludes that LTL shortening may be associated with a high risk of severe conditions and metabolic comorbidities in patients with psoriasis. Increased oxidative damage in telomeres may subsequently lead to the shortening of telomere length in these patients. These findings suggest that LTL is a good candidate biomarker for predicting the risk of poor prognosis in patients with psoriasis. Further studies with larger patient cohorts are required to validate the results of this study. The limitations of this study include the small sample size and lack of telomerase activity in the study groups.

This study complied with the Declaration of Helsinki. This study was approved by the Ethics Committees of Kuwait University and the Ministry of Health (State of Kuwait) (2016/496). Informed consent was obtained from all the participants.

The authors declare no conflicts of interest.

This study did not receive any external funding.

Conceptualization, methodology, and resources: Materah Salem Alwehaidah. Investigation, formal analysis, and writing – original draft, review, and editing: Materah Salem Alwehaidah and Moiz Bakhiet.

All data generated or analyzed during this study are included in this article. Further inquiries can be directed to the corresponding authors.