Frequency of Genotypes and Allelic Polymorphisms of Vitamin D Receptor in Egyptian Psoriatic Patients and Their Association With Disease Severity, Immune Modulation of IL-22 Levels and The Response to Topical Calcipotriol Treatment: A Case Control Study.

Objective: This study was performed to determine the genotype and allelic frequencies (polymorphisms) of the four genes of vitamin D receptor (VDR) among Egyptian psoriatic patients and healthy controls to explore their association with disease severity (PASI) score and immune modulation of IL-22 cytokine and to predict the response to topical calcipotriol treatment. Patients and
Methods: The frequencies of the four VDR gene polymorphisms (FokI, ApaI, TaqI, and BsmI) in blood samples of 51 adult Egyptian patients with psoriasis vulgaris and 50 healthy controls were evaluated using restriction fragment length polymorphism (RFLP)-PCR. Serum levels of IL-22 were measured by ELISA.
Results: The most frequent genotype (wild) in the studied patients was Apa1; AA (88.2%) followed by Fok1; FF (47.1%) and Taq1; TT (47%), while Bsm1; BB genotype was (27.7%). The most frequent allele polymorphisms either in one allele (Bb) or both alleles (bb) in psoriatic patients were 72.5%, followed by Ff, ff (52.9%) and Tt, tt (52.9%). The less frequent allelic polymorphism was Aa, aa (27.7%). Insignificant differences in the frequency of genotype (wild) and allelic polymorphisms were detected between patients and controls (P > 0.05). A significantly higher serum concentration of IL-22 (ng/mL) was detected in patients than controls (P = 0.001). Further, 66.6% of patients displayed a clinical response, while 33.4% were non-responders. A significantly higher expression of TaqI polymorphism was detected in (100%) of non-responders (P < 0.001), which was also correlated with disease severity (r = 0.515, P < 0.01).
Conclusion: These results suggest that the VDR TaqI polymorphism is the only gene correlated to psoriasis susceptibility in the Egyptian population, and affects the response to topical calcipotriol treatment but does not affect IL-22 immune modulation. Copyright:

Introduction

Psoriasis is a chronic inflammatory disease in which genetic, environmental, and immunological variables interact to develop synergy. Hyper-proliferation of keratinocytes is characteristic of the disease's pathophysiology, which is triggered by proinflammatory cytokines and leads to psoriatic lesions.[1]

It has been proved that 1,25-dihydroxyvitamin D3 analog (calcipotriol) has antiproliferative activities and is an effective therapy for psoriasis, targeting tissues through binding to its ligand, vitamin D receptor (VDR). This interaction inhibits the proliferation of keratinocytes.[2] However, the response to this treatment is variable in patients with psoriasis as some patients show partial resistance.[3]

VDR gene is located on chromosome 12q13. Four genotypes were recognized,[45] and four polymorphic sites (alleles) may occur in VDR gene. The protein synthesis start codon polymorphism is FokI (F, f), which is located in exon 2, TaqI (T, t) is located in exon 9, while BsmI (B, b) and ApaI (A, a) both are located in intron 8.[6]

Pilot studies to evaluate the clinical effectiveness of topical and oral calcipotriol treatment and its immunomodulatory effects on immune responses have been performed.[78] VDR is expressed by lymphocytes, monocytes, macrophages, and dendritic cells, among other immune cells.[9] Modulation of gene transcription of T-cell differentiation and proliferation occurs when calcipotriol binds to VDR.[10]

Several studies indicated that Th-1, Th-17, and Th-22 inflammatory responses to self-antigen have been found to be associated with the pathogenesis of psoriasis. They play a critical role in the hyperproliferation of keratinocytes, which results from chronic abnormal activation of the local immune cells. Production of increased amounts of cytokines and chemokines induces the recruitment and infiltration of leucocytes into the skin to create chronic inflammation.[11] Th-22 cells produce IL-22, IL-26, and IL-13, of which IL-22 is the most important inflammatory cytokine. IL-22 is present in large quantities in psoriatic lesions. Its receptor is present on epithelial/epithelioid cells that contributes to the formation of the psoriatic plaques.[1213]

Furthermore, genetic changes in psoriasis have been investigated to explore a possible association between the disease and the polymorphisms of the VDR gene which may result in keratinocyte activation.[1415]

Objective: This study was performed to identify the genotype and allelic frequencies (polymorphisms) of the four genes (FokI, ApaI, TaqI, and BsmI) of VDR among adult Egyptian patients with psoriasis vulgaris and healthy controls to explore their association with disease severity (PASI) score, immune modulation of IL-22 cytokine levels, and to predict the clinical response to topical calcipotriol treatment.

The Studied Population

Sample size

The sample size of the studied population was calculated using the (G power software). As regarding the primary outcome (the level of vitamin D in psoriatic patients), it was found that 50 participants per group were an appropriate sample size for the study with a total sample size of 100 participants. The power is 80%, α error probability = 0.05, and the effect size = 0.57. The magnitude of the effect to be detected was estimated as the mean and standard deviation of the variable of interest and obtained from the scientific literature.

Patients and controls

Fifty-one adult psoriasis vulgaris patients were recruited from Al-Zahraa University Hospital's Dermatology Department in Cairo, Egypt. There were 23 females and 28 males, ranging in age from 17 to 75 years. All patients were subjected to history taking of type treatment, clinical examination of disease manifestations extend of skin lesions, and the disease severity as expressed by PASI score.[16] The study also included 50 age and sex matched control subjects. VDR genotype and allelic polymorphisms, immune modulation of IL-22 cytokine, and levels of serum vitamin D were examined in those selected Egyptian patients.

According to disease severity, 18 patients were found to have mild disease. Their PASI score was ≤10. They were selected and treated with topical calcipotriol ointment 50 ug/g applied twice daily for 6 weeks as a thin layer to the affected skin. Further, the clinical response to treatment as measuring PASI score was recorded. Patients were classified into two groups: responders where the improvement was more than 50% and non-responders where the improvement was less than 50%.[17]

Each patient and control subject signed an informed written consent form. The study was approved by the university's ethics committee and this research was carried out in accordance with the principles of the Declaration of Helsinki.

Samples

Each patient and control subject had 3 mL of venous blood collected in tubes containing anticoagulants for DNA extraction. In addition, 2 mL was collected, sera were separated, and stored at −20°C till use in the measurement of IL-22 and vitamin D levels by ELISA.

Methods

Score on the Psoriasis Area and Severity Index (PASI): The severity of the disease was determined using PASI score.[16]

Genotyping of vitamin D receptor

Extraction of DNA:

DNA was extracted from the blood of patients and controls using the Gene Jet genomic DNA purification kit (Thermo Scientific) according to the manufacturer's instructions. DNA extracts were stored at −70°C till use.

Polymorphism assessment:

The four VDR gene polymorphisms (FokI, TaqI, ApaI, and BsmI) were assessed. FokI C>T(rs2228570) and TaqI T>C(rs731236) are polymorphic sites present on the coding sequence, and BsmI A>G(rs1544410) and ApaI G>T(rs7975232) are polymorphic sites present on the last intron.

The restriction fragment length polymorphism (REFLP) assay was performed after the polymerase chain reaction (PCR) amplification.

PCR amplification: A total volume of 25 μL containing Dream Taq Green PCR Master Mix (Thermo Scientific), 50 ng DNA sample, 100 μM dNTPs, 5 pmol of each primer, 0.75 mM MgCl2, 1× PCR buffer, and 0.5 U of Taq DNA. Three sets of primers were utilized, and their sequences were designed by using the NCBI Primer-Blast tool (http://www.ncbi.nlm.nih.gov/tools/primer-blast/) [Table 1]. A programmable thermal cycler Biometra, Germany was used for amplification.

Table 1

Polymerase chain reaction (PCR) primer sequences for detection of VDR polymorphisms

Genotype (SNPs)	Forward primer	Reverse primer	Specific PCR product (bp)
FokI	5′-TGCAGCCTTC ACAGGTCATA-3′	5′-GGCCTGCTTG CTGTTCTTAC-3′	157
TaqI,	5′-ACGTCTGCAG	5′-TCACCGGTCA	211
ApaI	TGTGTTGGAC-3′′	GCAGTCATAG-3′
BsmI	5′-CAGTTCACGC	5′-ACCTGAAGGG	236
	AAGAGCAGAG-3′	AGACGTAGCA- 3′

SNPs, single nucleotide polymorphisms

PCR cycling condition: Forty consecutive cycles, denaturation took 30 s at 95°C, annealing took 30 s at 61°C, and extension took 1 min at 72°C [Table 1].

PCR-Restriction Fragment Length Polymorphism (REFLP) for determination of VDR polymorphisms: Four restriction enzymes (Fast Digest FokI, TaqI, ApaI and BsmI; Thermo Scientific) were used for each sample to digest the PCR products; four separate tubes were prepared. Tubes containing ApaI restriction endonuclease enzyme were incubated at 37°C for 2 h, tubes with TaqI were incubated at 65°C for 30 min, tubes with and FokI were incubated at 37°C for 1 h. For each run, a negative control was included, containing all the reagents except the restriction enzyme. A total reaction volume of 30 uL was used, with 10 uL PCR products, 1 uL restriction enzyme, 5 uL buffer, and 14 uL sterile water. The digested products were run on a 2% agarose gel containing ethidium bromide and then visualized under UV light.

Measurement of IL-22 cytokine and vitamin D levels in sera by ELISA

Serum IL-22 levels were measured using ELISA Basic kit provided by MAB TECH, (Sweden Catalog #3475-1A-6). The procedure was performed according to manufactural instructions.

Vitamin D levels in sera were quantified using an ELISA kit following the manufacturer's protocols (DRG, International Inc., Springfield Township, NJ, USA).

Statistical analysis

SPSS Version 25.0 was used for statistical analysis (IBM Corp. Released 2017), IBM Armonk, NY: IBM Corp. USA. Quantitative variables were expressed as mean ± standard deviation; significant differences were determined using the t-test. Qualitative variables of VDR polymorphisms were expressed as numbers and percentages or frequencies using χ2 test. P < 0.05 was considered statistically significant.

Results

Table 1 lists the primers that were used in PCR.

Table 2 summarizes the results of (PCR-REFLP) before and after restriction enzyme digestion. The full PCR product for FokI was 157 bp in length; the restriction site was split into two bands of 121 and 36 bp. The full PCR product for TaqI was 211 bp in length; the restriction site was split into two bands of 172 and 39 bp. The full PCR product for ApaI was 211 bp in length; the restriction site was split into two bands of 121 and 90 bp, The full PCR product for BsmI was 236 bp in length; the restriction site was split into two bands of 197and 39 bp.

Table 2

Polymerase chain reaction (PCR) products digested by four restriction enzymes and the digested fragments

Enzyme	Interpretation	PCR product (bp) Baseline After digestion
FokI	Presence of restriction site (f)	157 121 and 36
	Absence of restriction site (F)	157
TaqI	Presence of restriction site (t)	211 172 and 39
	Absence of restriction site (T)	211
ApaI	Presence of restriction site (a)	211 121 and 90
	Absence of restriction site (A)	211
BsmI	Presence of restriction site (b)	236 197 and 39
	Absence of restriction site (B)	236

Genotypes were defined by capital letters in the absence of the restriction site (F, T, A, B) respectively) and small letters if the restriction site was present (f, t, a, b) respectively. Accession No. of FokI gene (NM001017535.2), ApaI and TaqI (NM 001374662.1) and BsmI (AY342401.1)

Demographic characteristics of psoriatic patients and controls

Table 3 summarizes the demographic characteristics of the patients and controls. The average age of the psoriatic patients and controls were (45.60 ± 15.00) and (38.40 ± 10.20) years, respectively. The patient group comprised 54.9% males and 45.1% females, while the healthy control group included 58% males and 42% females. There were no significant differences between the psoriatic patients and the controls (P > 0.05). The mean value of the disease duration was 7.75 ± 7.20 years. The extent of skin lesion ranged from 2% to 90%. The PASI score ranged from 1.4 to 30 and a mean of 11.20 ± 6.60.

Table 3

General characteristics of psoriatic patients and controls

Studied group Item	Psoriatic Patients n=51 No. (%)	Controls n=50 No. (%)
Sex (%):
Male	28 (54.9%)	29 (58%)
female	23 (45.1%)	21 (42%)
Age (Years):
Range	17-75	23-58
Mean±SD	45.6±15.0	38.4±10.2
Previous treatment:
Topical calcipotriol	18 (35.3%)
Other drugs (methotrexate, topical corticosteroid, emollient)	32 (62.7%)
No treatment	1 (2%)
Duration of psoriasis:
Range	(1 week-23 years)
Mean±SD (years)	7.75±7.2
PASI before:
Range	(1.4-30)
Mean±SD	11.2±6.6
Extent:
Range	(2%-90%)
Mean±SD	36% ± 28%

Frequency of genotypes (wild type) with absence of polymorphisms

The most common genotype (wild) in the studied Egyptian patients was Apa1; AA (88.2%) followed by Fok1; FF (47.1%), and Taq1; TT (47%). Bsm1; BB is the least genotype (27.7%). The same results were found in controls; the most common genotype was AA (88%) followed by FF (62%) and TT (48%). BB is also the least genotype (28%). There were no significant differences in the frequency of genotype (wild) between patient and control groups (P > 0.05) [Table 4].

Table 4

Gene and allelic distribution among patients and controls

Studied group Item	Cases (51)		Controls (50)		X 2	P
	No.	%	No.	%
Fokl
FF	24	47.1	31	0.2	0.32
Ff	24	47.1	15	30.0
ff	3	5.9	4	8.0
Bsml
BB	14	27.5	14	28.0	2.27	0.32
Bb	32	62.7	26	52.0	5
bb	5	9.8	10	20.0
Apal
AA	45	88.2	44	88.0	4.00	0.135
Aa	3	5.9	0	0.0
aa	3	5.9	6	12.0
Taql
TT	24	47.0	24	48.0	0.03	0.98
Tt	14	27.5	14	28.0
tt	13	25.5	12	24.0

The most common genotype (wild) in the studied Egyptian patients was Apa1; AA (88.2%). Bsm1; BB is the least genotype (27.7%). Insignificant differences in the frequency of genotype (wild) between patient and control groups (P>0.05)

Frequency of genotypes with presence of allelic polymorphisms

The most frequent allele polymorphisms either in one allele or both alleles in psoriatic patients were {Bb + bb (72.5%)} followed by {Ff + ff (52.9%)} and {Tt + tt (52.9%)}. The less frequent allelic polymorphism was {Aa + aa (27.7%)}. In controls, the most frequent allele was {Bb + bb (72%)}, followed by {Tt + tt (52%}) and (Ff + ff (38%)}. The less frequent allelic polymorphism was {Aa + aa (12%)}. However, insignificant differences were detected between patients and controls in VDR allelic polymorphisms (P > 0.05) [Table 4].

Serum concentrations of Vitamin D and IL-22 cytokine in psoriatic patients and controls

The mean values of vitamin D (ng/mL) in psoriatic patients and controls were 14.4 ± 4.5 and 25.6 ± 9.7, respectively. Serum concentrations of vitamin D in patients showed a highly significant reduction than controls (P = 0.001), while serum concentrations of IL-22 (ng/mL) in patients was significantly higher in psoriatic patients (104.5 ± 40.9) versus controls (27.7 ± 6.9), (P = 0.001) [Figure 1].

Figure 1

Box plot of serum concentrations of IL-22 cytokine and vitamin D in psoriatic patients and controls

Effect of topical calcipotriol treatment

Among 51 psoriasis vulgaris patients, 18 (35.3%) mild psoriatic patients were found to have a PASI score of <10 and treated only with topical calcipotriol ointment. The response to treatment was evaluated by PASI score after 6 weeks. The mean value of PASI score before treatment was 6.0 ± 0.97 and significantly reduced to 2.7 ± 2.4 (P < 0.001) after treatment [Figure 2]. Among 18 patients, 12 patients (66.6%) displayed response and designated responders while 6 (33.4%) were non-responders.

Figure 2

Effect of topical Calcipotriol treatment Significantly reduction in PASI score after treatment (P<0.001)

Distribution of VDR polymorphisms among topical Calcipotriol responders and non- responders

Patients who expressed Bsm1 {(Bb + bb) gene polymorphism had a higher response rate to calcipotriol treatment than patients with other polymorphisms as it is expressed in (91.7%), followed by Apa1 (Aa + aa) (33.3%) and Fok1 (Ff + ff) (25%). No polymorphisms were detected in Taq1 (Tt + tt) among responders.

In contrast, all non-responders (100%) expressed polymorphisms in Taq (Tt + tt) genotype followed by Bsm1 {Bb + bb (66.7%) and Fok1 {Ff + ff (50%)}. No polymorphisms were detected in Apa {Aa + aa}. A significant difference in Taq polymorphism was found between responders and non-responders (P < 0.001) [Table 5].

Table 5

Distribution of VDR polymorphisms among Calcipotriol responders and non-responders

Studied group Item	Responders (12)		Non-Responders (6)		X 2	P
	No.	%	No.	%
Fokl
+ve polymorphism (Ff &ff)	3	25.0	3	50.0	0.3	0.6
-ve polymorphism (FF)	9	75.0	3	50.0
Bsml
+ve polymorphism (Bb &bb)	11	91.7	4	66.7	0.5	0.5
-ve polymorphism (BB)	1	8.3	2	33.3
Apal	4	33.3	0	0.0	1.0	0.3
+ve polymorphism (Aa &aa)	8	66.7	6	100
- ve polymorphism (AA)
Taql	0	0.0	6	100.0	13.0	0.00*
+ve polymorphism (Tt &tt)	12	100.0	0	0.0
- ve polymorphism (TT)

Patients who were positive for Bsm1 (Bb & bb) gene polymorphisms had a higher response rate (91.7%) to Calcipotriol treatment. All non-responders (100%) were positive for Taq (Tt & tt) gene polymorphism. A significant difference in Taq polymorphisms was found between responders and non-responders (P<0.001)

Vitamin D and IL-22 concentrations (ng/ml) in responders and non-responders

Both responders (16.0 ± 3.9) and non-responders (11.9 ± 3.8) had lower levels of vitamin D less than controls (25.6 ± 9.7). However, responders had significantly higher levels of vitamin D than non-responders (P = 0.04). Although both responders (117.5 ± 41.5) and non-responders (91.2 ± 39) had higher levels of IL-22 than controls (27.7 ± 6.9), the difference between them was not significant (P = 0.2) [Table 6 and Figure 2].

Table 6

The mean levels of vitamin D and IL-22 in responders and non-responders

Studied group Item	Responders (12)	Non-responders (6)	t- test	P
Vit. D	16.0±3.9	11.9±3.8	2.2	0.04*
Mean±SD
IL 22	117.5±41.5	91.2±39.0	1.3	0.2
Mean±SD

Both responders and non-responders have lower levels of vitamin D less than controls. Responders have significant higher levels of vitamin D than non-responders (P=0.04). Both responders and no-responders have higher levels of IL-22 than controls, but the difference between them was not significant

Correlation study

Correlation coefficient study revealed insignificant correlations between VDR polymorphisms and any of the clinical parameters of all psoriatic patients. A positive correlation only was detected between severity of the disease (PASI) score and Taq polymorphism (r = 0.515, P < 0.01) [Figure 3].

Figure 3

Correlation coefficient between disease severity (PASI) score and Taq1 gene polymorphisms. There is a positive correlation between PASI score and Taql polymorphism (r=0.515, P<0.01)

Discussion

VDR genotyping and allelic polymorphisms in psoriasis had been investigated in different populations. However, the results were controversial.[18]

The current study revealed that the most frequent genotype (wild) in the studied Egyptian patients was Apa1; AA (88.2%), followed by Fok1; FF (47.1%) and Taq1; TT (47%). Bsm1; BB was the least frequent genotype (27.7%). Insignificant differences in the frequency of genotypes (wild) were revealed between patients and controls (P > 0.05) as the most common genotype was AA (88%), followed by FF (62%) and TT (48%). BB was also the least common genotype (28%).

In addition, the most frequent allele polymorphisms either in one allele or both alleles in psoriatic patients were {Bb + bb (72.5%)} followed by {Ff + ff (52.9%)} and {Tt + tt (52.9%)}. The less frequent allelic polymorphism was {Aa + aa (27.7%)}. The same results were detected in controls. Insignificant differences were detected between patients and controls in VDR allelic polymorphisms (P > 0.05).

In accordance with our findings, Zuel-Fakkar et al. (2010)[19] in Egypt found no significant differences in ApaI and TaqI vitamin D receptor gene polymorphisms between patients and controls. However, they reported that the Aa and Tt genotypes were the most prominent in both patient and control groups. The same results were also found in Turkish and Japanese populations.[620]

In contrast to our results, Korean researchers showed that there were significant differences in VDR genotype and allele frequencies between normal subjects and psoriasis patients, especially at early onset, and the aa genotype was most frequently in both patients and controls. They suggested that disequilibrium in VDR gene expression could be a risk factor for the occurrence of psoriasis. The contrast between those earlier results and ours could be explained by the size of the sampled population as our sample is relatively small and has different geographic regions.[6]

The multiplex SNaPshot method was used by Zhou et al.[21] in China to genotype psoriatic patients and controls for five common VDR gene variants (ApaI, TaqI, BsmI, FokI, and Cdx2). They also reported that psoriasis patients had a significantly higher frequency of ApaI (rs7975232) allele A than the control group (27.8% vs. 22.1%), and that allele A showed 1.35-fold increased chance of acquiring psoriasis. However, BsmI/ApaI/TaqI haplotype GCT presented to a smaller level extent in psoriasis patients in comparison with control patients (72.2% vs. 77.9%).

It was suggested that the polymorphic regions of the VDR gene may be related to the responsiveness to calcipotriol treatment. In the present study, topical calcipotriol ointment for 6 weeks displayed response in 66.6% of patients while 33.3% were non-responders, as indicated by a significant reduction in PASI scores. Responders have no polymorphisms in Taq1 (Tt + tt) or even have polymorphisms in Bsm1 {(Bb + bb) genotype (91.7%), Apa1 (Aa + aa) (33.3%) and Fok1 (Ff + ff) (25%). However, all non-responders had a significantly higher expression of TaqI polymorphism, which also correlated with disease severity of PASI score.

However, the impact of VDR polymorphisms in forecasting clinical response to topical calcipotriol was determined only in a few studies. Previously,[22] found that F and T alleles were positively related with calcipotriol response in patients of psoriasis.

In agreement with our results, Turkish researchers observed that the TT genotype and T allele were significantly more frequent in non-responders. They explained that the non-responsiveness to calcipotriol might be related to the type and severity of psoriasis disease.[6]

It was suggested that polymorphisms and haplotypes in the VDR gene may explain the differences in response to vitamin D therapy and that vitamin D deficiency is one of the independent major factors for psoriasis.[23] In the present study, serum concentrations of vitamin D revealed a highly significant reduction in psoriatic patients than controls.[15] stated that there is linkage between vitamin D deficiency and increased incidence of developing psoriasis. The most likely reason is that psoriasis patients are less likely to reveal their skin to sunshine, which is an important source of vitamin D.[24]

Our results confirmed this data as non-responders to topical calcipotriol treatment had significantly lower levels of vitamin D than responders. In general, monocytes/macrophages release 1,25 (OH) 2D, which causes a major shift in immunological status from proinflammatory to tolerogenic, suppresses T lymphocyte proliferation, and affects cytokine production. It encourages a shift from TH1, TH17, and TH22 inflammatory response to TH2 immune profile, resulting in hyperproliferation of keratinocytes.[11]

In contrast, the present work revealed a highly significant elevation in serum concentrations of IL-22 in all psoriatic patients and in both responders and non-responders to treatment than controls. However, the difference between responders and non-responders was not significant. This result was explained by[25] who stated that IL-22 cytokine was produced mainly by CD4+ T-cell populations. Its function is interplay between immune and epithelial cells; thus, it contributes to inflammation and epithelial remodeling of the psoriatic skin (Perera et al.; 2014).[26] In addition, Haneya A. A. Anani et al. (2017)[27] showed that the high level of IL-22 in psoriatic skin lesion and rats with genetic deficiency of IL-22 natural inhibitors displayed worsened disease that associated with increasing the expression of IL-22–inducible antimicrobial peptides.

Conclusion: These results suggest that the VDR TaqI polymorphism is the only gene correlated to psoriasis susceptibility in the Egyptian population and affects the response to topical Calcipotriol treatment but does not affect IL-22 immune modulation.

Recommendations

Systemic immunosuppressive or phototherapy should be administered to improve the clinical response in non-responders to topical treatment.

Limitation of the study

The studied number of patients and controls is relatively small. When studying the polymorphism in future studies, the number should be increased.

Declaration of patient consent

The authors certify that they have obtained all appropriate patient consent forms. In the form, the patient(s) has/have given his/her/their consent for his/her/their images and other clinical information to be reported in the journal. The patients understand that their names and initials will not be published and due efforts will be made to conceal their identity, but anonymity cannot be guaranteed.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.