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Propensity for DNA Damage in Psoriasis Patients Genotyped for Two Candidate Genes | OMICS International
ISSN: 2157-2518
Journal of Carcinogenesis & Mutagenesis

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Propensity for DNA Damage in Psoriasis Patients Genotyped for Two Candidate Genes

G. Gandhi1*, Premjot Singh Girgila2, Ramesh K. Aggarwal3 and Brinderjit Singh Buttar1

1Department of Human Genetics, Guru Nanak Dev University, Amritsar, Punjab, India

2Formerly at Skin and T.B. Ward, Sri Guru Ram Das Charitable Hospital, Amritsar, Punjab, India

3Molecular Genetics Lab., Centre for Cellular and Molecular Biology, Hyderabad, India

*Corresponding Author:
Dr. G. Gandhi
Department of Human Genetics
Guru Nanak Dev University
Amritsar 143 005, India
Tel: +91-183-2258802-09 Extn. 3444
Fax: +91-183-2258820
Email: [email protected]

Received date: November 13, 2010; Accepted date: December 04, 2010; Published date: December 06, 2010

Citation: Gandhi G, Girgila PS, Aggarwal RK, Buttar BS (2010) Propensity for DNA Damage in Psoriasis Patients Genotyped for Two Candidate Genes. J Carcinogene Mutagene 1:112 doi: 10.4172/2157-2518.1000112

Copyright: © 2010 Gandhi G, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

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Studies assessing genetic damage and its association with disease-candidate genes in patients belonging to geographically distinct populations are scanty. The present study evaluated DNA damage using the alkaline Single Cell Gel Electrophoresis assay in peripheral blood leukocytes of Psoriasis Punjabi Patients on systemic-topical therapy who had been genotyped for two disease-candidate genes (HLA-C, human leukocyte antigen and the coiled-coil alpha-helical rod protein 1(CCHCR1). Genetic damage was disease gene-influenced as homozygous mutants for CCHCR1 Exon 4 site 386* (C→T) and heterozygous mutants for 404* (C→T) alleles had significantly more damage (p<0.05) compared to respective homozygous wild types. The arginine to tryptophan substitution alters the protein, triggering keratinocyte proliferation and probably inflammation/ oxidative stress. This along with drug-treatment probably caused the observed DNA damage. Population sub-groups had no within group differences but larger sizes can explore this possibility. Studies of this type can provide disease-gene-damage prone information for exploring DNA- safe therapeutics.


Genetic damage; Gene association; Population subgroups


Studies for associations between polymorphisms of candidate disease genes and genetic damage in geographically defined populations have not come to attention. Such studies can explore ethnic and racial differences in disease as well as propensity for DNA damage since the latter is an early indicator of carcinogenesis. Assessment of patients for genetic damage can assist in its management. Psoriasis, an immune-mediated inflammatory skin disease requiring long term treatment, affecting 2–3% of the population [1] with varying ethnicfrequency [2], mediated via genetic-environmental factors, causes keratinocyte hyperproliferation, inflammatory response [3] with an impaired oxidant/antioxidant status [4-6] which is damaging to proteins, lipids and DNA. Patients with/without various treatment modalities have been assessed for genetic damage [7-11] but none on both, systemic and topical treatments which is a regular, local prescription. Therefore DNA damage was investigated in peripheral blood leukocytes (PBL) of patients applying coal tar ointment and taking cyclosporine and/or Methotrexate using the alkaline single cell gel electrophoresis (SCGE) assay which assesses DNA strand breakage at cell level [12]. Patients were genotyped (unpublished work) for polymorphisms in two candidate genes: coiled-coil alpha-helical rod (CCHCR1) protein 1 (exons 4 and 10) and HLA-C region (SNPn7 and SNPn9) as their proteins are involved in manifestation of Psoriasis. CCHCR1 protein is differently expressed in lesional psoriatic skin with a role in keratinocyte proliferation [13] and HLA-C protein levels are increased in plaque psoriasis skin [14].

Questions Addressed

As propensity for genetic disease is ethnicity-dependent and as susceptibility for genomic insult has an association with metabolic genotypes, there could also be an association underlying disease gene-specificity and genetic damage. With this hypothesis and in view of cited literature, Psoriasis patients on systemic and topical therapy, already genotyped for some polymorphisms of CCHCR1 gene and HLA-C region, were assessed for DNA damage (pre-cancerous lesions) in order to correlate the level of genetic damage, if any, with their genotypic status, population sub-group besides routine variables. To the best of our knowledge, no such studies have been reported, at least not in Psoriasis patients from this region.

Experimental Design

Patients and healthy, matched controls participated after voluntary, written informed consent and study’s institutional ethical clearance. Disease history, demographic information and pedigree were recorded on a questionnaire. PBL from genotyped patients were processed for DNA damage by alkaline SCGE assay [15] except use of local chemicals and silver staining. Under electrophoresis, breaks in the super coiled DNA migrate towards the anode and appear as a comet indicative of DNA damage. Coded slides were visually scored at 400X. DNA migration was measured using a calibrated ocular micrometer and cells were graded into categories based on tail length. Arbitrary score, damage frequency (DF) and damage index (DI) were calculated [16] as mean ± S.E.M. Mann-Whitney U test was applied for significance of DNA damage. Chi-square (χ2) test compared attributes of patients and controls and their cells in different damage categories. Regression analysis, analysis of variance and odds ratio at 95% confidence interval were performed for confounders of DNA damage. Values were taken significant at p≤0.05, p<0.01and p<0.001. All analysis was done using SPSS (version 10.0).


Characteristics of patients and matched controls are given in supplementary material (Table S1, Table S2). DNA migration length, DI and DF were significantly elevated (p<0.05, p<0001) in patients compared to controls (Table 1). Patients homozygous for CCHCR1 386* (C→T) mutant allele and those heterozygous for 404* (C→T) allele had significantly (p<0.05) more DNA damage than respective homozygous wild types. Homozygous mutants of these alleles in controls were lacking. Comet categories 1-3 were also significantly higher (p<0.05; Table S3). Multiple regression analysis revealed methotrexate-coal tar-cyclosporine combination (p=0.041), treatment time (p=0.045) and CCHCR1 386* (C→T) allele association (p=0.021) with DNA damage in patients. Though Jat Sikh patients had more damage, there were no significant endogamous-group differences and damage in each group was significant from respective controls: Jat Sikhs at p≤0.001, Brahmins at p≤0.01, Ramgarhia and Majbi Sikhs each at p≤0.05. Early onset patients (<40y, type I) had significant difference (p=0.004) compared to late on-set (≥40y, type II) but with no gender differences (Table S4). In vulgaris (p=0.000) and palmo-plantar (p=0.002) types, damage was higher from controls but not from each other (Table S5). Methotrexate (2.5mg/week), coal tar application (5.3%; once daily) with/without cyclosporine (25mg/week) for 2-27y caused significant damage (p<0.001) as did treatment duration but non-significantly (Table S6). Newly diagnosed patients (n=3) also had more damage compared to controls but less from treated group.

  Allele Genotype PATIENTS  
DI± S.E.M. (n) DF± S.E.M. (n) Mean  DNA migration length ± S.E.M. (n) DI± S.E.M. (n) DF± S.E.M. (n) $Mean  DNA migration length ± S.E.M. (n)
HLA-C region 22222* (G→A) GG 28.38***±1.79 (12) 55.23***±3.28 (12) 37.51***±1.56 (12) 4.88±1.28  (9) 11.77±2.59 (9) 9.38±1.94 (9)
GA 28.41***±1.94 (16) 55.29***±3.62 (16) 33.78***±2.60 (16) 6.75±2.56  (4) 13.50±5.12 (4) 12..29±4.36 (4)
AA 33.42*±2.12  (7) 62.85*±3.17  (7) 33.41*±3.49  (7) 6.00±1.00  (2) 12.00±2.00 (2) 9.07±0.68 (2)
22333* (A→G) AA 28.25**±1.95  (12) 54.83***±3.54  (12) 36.92***±1.58  (12) 10.66±5.10  (9) 11.33±2.78 (9) 8.79±2.17 (9)
AG 28.31***±2.06 (16) 55.12***±3.85 (16) 33.86***±2.77 (16) 6.75±2.56  (4) 13.50±5.12 (4) 12.29±4.36 (4)
GG 33.57*±1.70 (7) 62.85*±3.17 (7) 33.41*±3.49  (7) 6.00±1.00 (2) 12.00±2.00 (2) 9.07±0.68 (2)
24118* (T→C) TT 28.90***±2.01 (11) 56.18***±3.59 (11) 36.76***±1.72 (11) 9.00±3.60  (13) 11.07±2.28 (13) 8.69±1.77 (13)
TC 27.72±1.87  (18) 53.88±3.53  (18) 34.02±2.47 (18) 9.00±0.00 (1) 18.00±0.00 (1) 17.56±0.00 (1)
CC 34.83±1.88  (6) 65.33±2.34 (6) 33.66±4.12  (6) 9.00±0.00  (1) 18.00±0.00 (1) 15.79±0.00 (1)
CCHCR1 gene 386* (C→T) CC 26.09***ª±2.02 (11) 50.36***ª±3.51(11) 32.83***ª±2.38 (11) 9.28±3.32 (14) 9.28±3.32(14) 9.86±1.82(14)
CT 29.40±1.59 (21) 57.40±3.06(21) 36.31±2.11 (21) 5.00±0.00 (1) 10.00±0.00(1) 8.39±0.00(1)
TT 37.00 ª ±2.64(3) 66.66 ª ±1.33(3) 36.37 ª ±5.66(3) - - -
404* (C→T) CC 25.66*** b ±1.84  (9) 49.33*** b ±2.84 (9) 31.32***±2.60 ( 9) 9.28±3.32  (14) 12.14±2.21 (14) 9.86±1.82 (14)
CT 30.30 b ±1.70  (20) 59.20 b ±3.21 (20) 35.07±2.05  (20) 5.00±0.00 (1) 10.00±0.00 (1) 8.39±0.00 (1)
TT 31.50±3.41  (6) 58.66±5.35 (6) 39.24±3.85  (6) - - -
1364* (T→C) TT 28.01***±4.27 (10) 56.20***±5.92 (10) 38.14***±3.61 (10) 6.40±1.39 (10) 12.80±2.78 (10) 10.83±2.22(10)
TC 30.31***±1.47(17) 59.12***±2.57 (17) 34.64***±1.93 (17) 5.25±1.93 (4) 10.50±3.86(4) 7.63±3.25(4)
CC 25.60±2.37 (8) 10.00±0.00 ( 8) 30.00±4.23 (8) 50.00±0.00 (1) 10.00±0.00(1) 7.55±0.00(1)

Table 1: HLA-C and CCHCR1 genotypes and DNA damage in Psoriasis patients and controls.


DNA migration length, DF and DI reflect cellular events manifesting from DNA strand breaks. Propensity for genetic damage was disease gene-influenced as homozygous mutants for CCHCR1 Exon 4 site 386* (C→T) and heterozygous mutants for 404* (C→T) alleles had significantly more damage (p<0.05) compared to their respective homozygous wild types. The CCHCR1 protein plays a role in keratinocyte biology and in malignant transformation [17] and the CC to TT mutant change in its two SNPs could be altering the secondary structure from the wild type protein as tryptophan substitutes arginine [18] probably provoking an inflammatory/oxidative response and promoting DNA damage as observed in the mutant homozygous/ heterozygous patients but this requires confirmation. In Chinese and Finnish patients with these reported SNPs [18,19], genetic damage assessment can provide insights for disease gene- susceptibility and DNA damage. Punjab people are mostly Sikhs and Hindus by religion but belong to different endogamous groups. These patients had comparable damage but larger samples can explore damage susceptibility/resistance.

The observed DNA damage probably resulted from diseaseinduced oxidative stress with a potentiating effect of the therapeutics. The CCHCR1 allele product may too be modulating some aspects of DNA damage. Since genetic damage is prequel to neoplasia, increased risk in Psoriasis patients exists for developing cancer even in nontarget cells in addition to developing “systemic” co-morbidities [20] and basal cell carcinoma. To our knowledge this is a first study of its kind. Such population-specific disease-gene and genetic damage association studies can provide disease-damage susceptibility/ resistance information which can be useful for exploring targetspecific DNA- safe therapeutics.


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