ALL Metrics
-
Views
-
Downloads
Get PDF
Get XML
Cite
Export
Track
Study Protocol

Comparison of serum vitamin D level and vitamin D receptor gene FokI polymorphism in leprosy patients with and without trophic ulcers: protocol for a case-control study

[version 1; peer review: 1 approved with reservations]
PUBLISHED 09 Jan 2025
Author details Author details
OPEN PEER REVIEW
REVIEWER STATUS

This article is included in the Neglected Tropical Diseases collection.

Abstract

Background

Leprosy, or Hansen’s disease, is a chronic infectious disease that primarily affects the skin and peripheral nervous system, often leading to disabilities, including trophic ulcers (TU). The role of vitamin D and vitamin D receptor (VDR) gene polymorphisms in the development of TU among leprosy patients remain unclear.

Objective

This study aims to investigate the differences in serum 25-hydroxyvitamin D [25(OH)D] levels and the frequency of VDR gene FokI polymorphism between leprosy patients with and without TU, and to evaluate their association with the occurrence of TU.

Methods

This Institutional Review Board-approved, single-center, observational, analytic case-control study will enroll adult leprosy patients from the Dermatology and Venereology Clinic at Dr. Cipto Mangunkusumo Hospital in Jakarta, Indonesia. Participants will be divided into cases (patients with TU) and controls (patients without TU). Serum 25(OH)D levels will be measured using a chemiluminescence immunoassay, and FokI polymorphism detection is conducted through polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) methods. Statistical analysis will be performed to evaluate the differences and associations.

Results

Data collection commenced in March 2024 and was completed in September 2024. Data analysis and reporting are expected to be finalized by January 2025.

Discussion

The findings of this study will enhance the current understanding of the role of the vitamin D axis in the development of TU among leprosy patients. This could lead to new therapeutic strategies, such as vitamin D supplementation or personalized medicine based on genetic profiling, potentially reducing the burden of TU among leprosy patients.

Keywords

trophic ulcer, leprosy, vitamin D, vitamin D receptor gene polymorphisms

Introduction

Background

Leprosy, also known as Hansen’s disease (HD), is a chronic infectious disease caused by the bacteria Mycobacterium leprae and Mycobacterium lepromatosis. This disease primarily affects the skin and peripheral nervous system, leading to disabilities. Leprosy remains endemic in many parts of the world, with Brazil, India, and Indonesia collectively accounting for 80% of new cases globally.1 In 2019, the Indonesian Ministry of Health reported 17,439 new cases of HD, while in 2020, only 11,173 new cases were identified.2,3 The COVID-19 pandemic has impacted early detection efforts due to social restrictions and the reallocation of healthcare resources, potentially increasing the incidence of disabilities resulting from delayed diagnosis and treatment.4

Peripheral nerve damage secondary to leprosy can lead to various types of disabilities. Ulcers are a common complication and are classified as grade 2 disabilities.5 The rate of grade 2 disabilities is a key indicator in the World Health Organization’s (WHO) 2021-2030 leprosy eradication strategy. In 2021, the rate of grade 2 disabilities in Indonesia was 2.48 cases per 1,000,000 people, whereas the WHO’s 2030 target is 0.12 cases per 1,000,000 people.2 Disabilities associated with leprosy create social stigma and lead to significant economic losses, including healthcare costs and reduced productivity.6

Ulcers occur in 10–20% of HD patients and are mostly classified as trophic ulcers (TU).5 Most ulcers in HD patients occur in the lower extremities, particularly on the plantar surface of the feet.5,7 Static and dynamic deformities of the feet, coupled with reduced sensitivity, lead to recurring wounds that do not heal and become chronic ulcers.5,7 The occurrence of TU in leprosy patients can be influenced by several factors, including neuropathy, physical deformities, nutritional deficiencies, and other systemic comorbidities. In leprosy patients, ulcers are often difficult to treat and frequently recur. If not properly managed, ulcers can become secondarily infected and may even undergo malignant transformation.8

Vitamin D is a steroid hormone that plays a crucial role as an immune system modulator.9,10 Over the past decade, studies have reported the role of vitamin D in wound healing.9,1113 Vitamin D enhances the differentiation of fibroblasts and keratinocytes, mainly through the modulation of growth factors and cytokine production.14,15 In vivo, vitamin D is converted to its active form, 25-hydroxyvitamin D [25(OH)D], by the hepatic hydroxylase enzymes, which is an indicator of vitamin D status. In the innate immune system, 25(OH)D can trigger the production of antimicrobial peptides (AMPs) by monocytes and macrophages.10 Additionally, 25(OH)D reduces the production of proinflammatory cytokines and increases anti-inflammatory responses.16 The kidneys or injured tissues can convert 25(OH)D into 1,25-dihydroxy vitamin D3 or calcitriol, which regulates the expression of cathelicidin and defensins.15,17 These AMPs can kill intracellular bacteria and play a significant role in wound healing.10,15

The biological effects of vitamin D are mediated by the vitamin D receptor (VDR), which is encoded by the VDR gene on chromosome 12. Animal studies have reported that loss of VDR inhibits tissue re-epithelialization and inflammatory response in wound healing.18,19 Various single nucleotide polymorphisms (SNPs) can affect the structure and function of VDR. The FokI polymorphism (rs2228570), which involves a nucleotide substitution from thymine (T) to cytosine (C) at the start codon of the VDR gene, is the only polymorphism known to structurally alter the VDR protein. The mutant T allele (f allele) yields a receptor with a longer amino acid chain compared to the wild-type C allele (F allele).20 The F allele produces a VDR with more optimal gene transcription effects, thereby triggering increased immune responses and wound healing.20,21 A study in Medan, Indonesia reported that the frequency of homozygous mutant subjects for the FokI polymorphism was 19.2% in multibacillary HD patients and 13.7% in healthy household contacts of HD patients.22

Previous studies have shown that serum 25(OH)D levels in patients with other chronic ulcers (e.g., venous or diabetic ulcers) are decreased,23,24 and vitamin D supplementation can improve wound healing in these conditions.12,13 The role of vitamin D in leprosy is increasingly investigated, with previous studies reporting varied results.2527 However, to the best of our knowledge, no research has compared vitamin D levels and the polymorphisms of its receptor in leprosy patients with and without TU.

Aim

The aim of this study is to investigate whether vitamin D and the polymorphisms of its receptor (VDR) play a role in the development of TU in leprosy patients. The primary and secondary objectives of the study are displayed in Table 1.

Table 1. Primary and secondary objectives.

Objective typeDescription
Primary

  • To analyze the differences of 25-hydroxyvitamin D [25(OH)D] levels in leprosy patients with and without trophic ulcers.

  • To analyze the proportion difference of FokI polymorphism in leprosy patients with and without trophic ulcers.

  • To evaluate whether 25(OH)D levels and FokI polymorphism are associated with the occurrence of trophic ulcers in leprosy patients.

SecondaryTo determine whether there is any correlation between 25(OH)D levels and the severity of trophic ulcers.

Protocol

Study design

This study is a single-center, observational, analytic case-control study.

Site and population

Leprosy patients with and without TU who are attending the Dermatology and Venereology Clinic at Dr. Cipto Mangunkusumo Hospital in Jakarta, Indonesia will be recruited. Serum 25(OH)D levels will be analyzed at the Clinical Pathology Laboratory of the same hospital. Examination of the FokI polymorphism will be conducted at the Laboratory of the Medical Biology Department, Faculty of Medicine, Universitas Indonesia, Jakarta.

Case and control definitions

Both cases and controls include adult leprosy patients diagnosed according to the cardinal signs defined by WHO. Patients who are newly diagnosed, currently undergoing treatment, or have been released from treatment are eligible for inclusion. The inclusion and exclusion criteria are detailed in Table 2. The control group will consist of leprosy patients without TU, who meet the same inclusion and exclusion criteria as the case group.

Table 2. Inclusion and exclusion criteria.

CriteriaDescription
Inclusion

  • Patients willing to participate in the study by completing and signing an informed consent form.

  • Male or female patients aged ≥18 years old.

  • Patients who meet the World Health Organization (WHO) diagnostic criteria for leprosy, including those who are newly diagnosed, currently undergoing treatment, or have been released from treatment.

  • Leprosy patients with trophic ulcers (case group), adhering to the following criteria:

    • Ulcer duration of ≥4 weeks, with no history of sharp debridement or callus thinning during this period.

    • Wound depth not exceeding the subcutaneous layer.

    • Ulcer not secondarily infected or only mildly infected (characterized by erythema <2 cm around the ulcer, potential signs of edema, pain, minimal purulent secretions, or warmth on palpation).

Exclusion

  • Patients who have consumed vitamin D supplementation within the past 4 weeks.

  • Patients exhibiting signs and symptoms of leprosy reactions.

  • Patients with concurrent systemic infections, diabetes mellitus, severe liver dysfunction, end-stage kidney disease, malignancy, and autoimmune diseases.

Subject recruitment

Subjects will be recruited consecutively based on the eligibility criteria. All leprosy patients attending the Dermatology and Venereology Clinic of Dr. Cipto Mangunkusumo Hospital will be given the opportunity to participate in this study and will be screened according to the inclusion and exclusion criteria. Before further examination, each potential subject will be informed about the purpose and methods of the study, as well as the benefits and potential disadvantages of participating. We will explain to potential subjects that participation is voluntary and does not affect the management provided at the hospital. Written informed consent will be obtained from each subject.

Procedures and examinations

History taking and physical examination will be conducted in a structured manner following a standardized study data collection form. Data collected during anamnesis will include identity, sociodemographic history, leprosy history (duration, treatment, and reaction history), duration of sun exposure, smoking status, and ulcer duration. Physical examination will include vital signs, anthropometric measurements, dermatologic status of the ulcer, presence of deformities other than ulcers, and sensory function of the extremities. Sensory function examination will be assessed using 0.2-gram (blue) Semmes-Weinstein monofilaments for hands and the 2-gram (purple) monofilaments for feet.28

Clinical photographs of the subjects’ hands and feet are collected, clearly showing the ulcers if present. If ulcers occur on other areas of the extremities (e.g., knee or elbow), documentation will also be performed on these areas. Photos are taken using an iPhone 12 camera (12 megapixel, aperture size F1.6, focal length 26 mm, sensor size 1/2.55", pixel size 1.7 μm) from a distance of 15 cm. A blue cloth will be used as the background during photo taking.

The dimensions of the ulcer are measured using a digital caliper, with the ulcer area calculated by the multiplying the largest vertical and horizontal lengths that intersect perpendicularly. Ulcer depth will be qualitatively evaluated according to the Pressure Ulcer Scoring System (grades I to III). Ulcer severity will be assessed using the Pressure Ulcer Scale for Healing (PUSH) criteria, which encompasses ulcer area, exudate amount, and wound tissue type ( Table 3). In patients with multiple ulcers, the largest ulcer by area will be selected for reporting and further statistical analysis.

Table 3. Pressure Ulcer Scale for Healing (PUSH).

CharacteristicsScore Subscore
Length × Width (in cm2) 0
0
1
<0.3
2
0.3–0.6
3
0.7–1.0
4
1.1–2.0
5
2.1–3.0
6
3.1–4.0
7
4.1–8.0
8
8.1–12.0
9
12.1–24.0
10
>24.0
Exudate amount 0
None
1
Light
2
Moderate
3
Heavy
Tissue type 0
Closed
1
Epithelial Tissue
2
Granulation Tissue
3
Slough
4
Necrotic tissue
Total:

Laboratory prodecures

Blood samples totaling 6 mL will be collected from each subjects into plain (red-capped) and K3 EDTA (purple-capped) tubes and temporarily stored in a cool box at 2–8°C. The blood samples will be sent to the laboratories within 2 hours of collection.

25(OH)D Level quantification

Blood collected in a plain tube will be analyzed for 25(OH)D levels using the Architect 25(OH)D vitamin kit (Abbott Diagnostics, Lake Forest, IL, USA) based on the chemiluminescence immunoassay method. Vitamin D status will be classified under four categories: severe deficiency (<10 ng/mL), deficiency (10-20 ng/mL), insufficiency (20-30 ng/mL), and sufficiency (>30 ng/mL).29

FokI polymorphism detection

The anticoagulated blood will be used for FokI polymorphism detection, which consists of DNA extraction, subsequent amplification using polymerase chain reaction (PCR), and polymorphism detection using the restriction fragment length polymorphism (RFLP) method. DNA extraction from EDTA blood samples will be performed using the salting out method.30 Extracted genetic materials will be stored on a -80°C freezer for subsequent analysis.

Amplification of the genetic materials are performed using PCR. The primers for the reaction are based on a previous study by Soroush et al.,20 with a forward primer 5′-CACTGACTCTGGCTCTGACCGT-3′ and a reverse primer 5′-AACACCTTGCTTCTTCTCCCTCC-3′. Initial denaturation of the genetic material will be performed at 95°C for 5 minutes. Amplification will be conducted for 35 cycles with denaturation at 95°C for 30 seconds, annealing at 60°C for 30 seconds, and extension at 72°C for 30 seconds. The final extension will be performed at 72°C for 7 minutes, resulting in a nucleic acid product of 250 base pairs (bp) in length.

The RFLP will be conducted using the FokI restriction enzyme (New England Biolabs, Ipwich, MA, USA). After incubation, the results will be visualized with 2% agarose gel electrophoresis. The genetic material fractions that will be detected are 192 and 58 bp in homozygous ff genotype samples, 250 bp, 192 bp, and 58 bp in heterozygous Ff genotype samples, and an intact 250 bp fragment in FF genotype samples.

Sample size estimates

The calculation of sample size is carried out using a type I error probability of 0.05 and type II error of 0.20, corresponding to a power of 80%.

To test the difference in mean 25(OH)D levels, sample size calculation for the difference between the mean values of two independent groups is performed using the following formula31:

n1=n2=2[(z+zβ)s(x1x2)]2
where n1 = n2 = minimum sample size for each group; zα = z-score corresponding to the type I error probability, which is 1.96; zβ = z-score corresponding to the type II error probability (β = 0.20), which is 0.84); s = standard deviation; and (x1x2) = clinically significant mean difference. The clinically significant mean difference is determined to be 5 ng/mL. Since there has been no studies reporting the difference in 25(OH)D levels between leprosy groups with and without TU, the standard deviation value used is based on a previous study in general leprosy patients, which is 5.42 ng/mL.27 Thus, the sample size needed to detect a difference in 25(OH)D levels in leprosy patients with and without TU is 19 per group.

To test the effect of FokI polymorphism on the occurrence of TU in leprosy patients, the sample size for an unmatched case-control study is calculated using the formula31:

n1=n2=(zα2PQ+zβP1Q1+P2Q2)2(P1P2)2
where n1 = n2 = minimum sample size for each group; zα = 1.96; zβ = 0.84; P1 = estimated proportion of effect in the case group calculated using P1=OR×P2(1P2)+(OR×P2) ; OR = estimated odds ratio; P2 = proportion of effect in the control group; P = ½ (P1 + P2); and Q = 1 – P. The proportion of the homozygous ff genotype in leprosy patients based on a previous study by Lubis et al.22 is 19.2%. Assuming an estimated odds ratio (OR) of 3.9 as the effect size of FokI polymorphism on TU occurrence in leprosy patients, the formula corresponds to a sample size of 41 for each of the case and control groups.

Finally, to test the correlation between 25(OH)D levels and ulcer severity, sample size calculation for correlation is conducted using the following formula31:

n=[(z+zβ)0.5ln(1+r1r)]2+3
where n = minimum sample size for the group; zα = 1.96; zβ = 0.84; and r = estimated correlation coefficient. A moderate correlation (r = 0.5–0.7) is estimated between ulcer severity score and serum 25(OH)D levels. Thus, setting the correlation coefficient at 0.5, the minimal sample size is 29 for this analysis. The overall sample size for this study is determined based on the largest minimum sample size required to test all outcomes, which is 41 for each group of leprosy with and without TU, totaling 82 subjects.

Data analysis

All data obtained from anamnesis, physical examinations, and laboratory tests are recorded in the research file. The collected data will be reviewed, coded, and then entered into a master table in Excel version 16 (Microsoft Corporation, Washington, USA). Data analysis will be conducted using STATA version 16.0 (StataCorp LLC, College Station, TX, USA) and the online software SNPStats (www.snpstats.net, Catalan Institute of Oncology, Spain).

Sociodemographic and clinical characteristics data will be processed descriptively and presented in tables to understand the distribution of data across groups. Clinical characteristics of TU will also be described and presented in a table. Data normality will be tested using the Kolmogorov-Smirnov test. Numerical data will be presented as mean and standard deviation (SD), or as median and interquartile range (IQR) if not normally distributed.

A significance level of p < 0.05 will be used throughout the study. The comparison of means between two groups will be conducted using the independent t-test for normally distributed data or the Mann-Whitney U test if the data are not normally distributed. Relationship between two categorical variables will be analyzed using the chi-square or Fisher’s exact test, as appropriate. Correlations between two numeric variables will be calculated using the Pearson’s correlation coefficient for normally distributed data or Spearman’s rank correlation coefficient if the data are not normally distributed.

The Hardy-Weinberg equilibrium will be assessed to determine if the observed genotype frequencies are consistent with those expected in a population for both case and control groups. Different genetic models (i.e. codominant, dominant. recessive, overdominant, and log-additive) for the FokI polymorphism will be evaluated. Both the Akaike information criterion (AIC) and the Bayesian information criterion (BIC) will be used to compare these models. The genetic model yielding the lowest AIC and BIC values will be considered the best-fitting model.

Finally, multivariate logistic regression analyses will be conducted to identify factors associated with the occurrence of TU in leprosy patients. Variables with a p-value < 0.25 in bivariate analysis will be included in the multivariate model. Adjusted odds ratios (ORs) and 95% confidence intervals (CIs) will be reported for the variables included in the final model.

Ethics and consent

This study protocol has received ethical clearance from the Health Research Ethics Committee –Faculty of Medicine Universitas Indonesia and Dr. Cipto Mangunkusumo Hospital (HREC FMUI-CMH), with the approval letter number KET-82/UN2.F1/ETIK/PPM.00.02/2024, dated January 15, 2024.

We will explain to potential subjects that participation is voluntary and does not affect the management provided at the hospital. Written informed consent will be obtained from each subject.

Dissemination

The results of the study will be disseminated through peer-reviewed publications and will be made available in an open-access format. Any significant changes to the protocol will be communicated directly to HREC FMUI-CMH. We also plan to publish any modifications made to this protocol during publication of the study results.

Study status

Subject recruitment, assessment, and laboratory work for 25(OH)D level measurement and FokI polymorphism detection have been completed. Formal data analysis is currently ongoing.

Discussion

Summary

This study aims to investigate the association between serum vitamin D levels and FokI polymorphism of VDR in leprosy patients, comparing those with and without TU. Leprosy often leads to peripheral nerve damage, with TU as a common complication. We hypothesize that vitamin D and its receptor polymorphisms, particularly FokI, might influence ulcer development in these patients.

Our study is a single-center, case-control study involving adult leprosy patients with and without TU. Serum vitamin D levels are quantified using chemiluminescence immunoassay, while the FokI polymorphism of the VDR gene is detected through PCR-RFLP method. The primary objectives are to analyze differences in 25(OH)D levels and the proportion of FokI polymorphism between the two groups, and to evaluate their association with TU occurrence. Secondary objectives include examining the correlation between vitamin D level and the severity of TU.

Perspective and clinical implications

Trophic ulcers may still occur in leprosy patients long after treatment completion. As the world approaches the leprosy post-elimination era, TU will continue to cause significant morbidity. Therefore, the study’s focus on the potential influence of vitamin D and its receptor polymorphisms on TU development in leprosy is of significant clinical importance.

If substantial evidence is found, this could pave the way for new treatment strategies, such as vitamin D supplementation or personalized therapies based on genetic profiles in leprosy patients. This could enhance patient outcomes, reduce the incidence of trophic ulcers, and consequently diminish the healthcare and social burdens associated with leprosy-related disabilities. Additionally, the findings could contribute to a better understanding of the role of the vitamin D axis in the pathophysiology of TU and other chronic ulcers in general.

Comments on this article Comments (0)

Version 1
VERSION 1 PUBLISHED 09 Jan 2025
Comment
Author details Author details
Competing interests
Grant information
Copyright
Download
 
Export To
metrics
Views Downloads
F1000Research - -
PubMed Central
Data from PMC are received and updated monthly.
- -
Citations
CITE
how to cite this article
Halim PA, Sirait SP, Miranda E et al. Comparison of serum vitamin D level and vitamin D receptor gene FokI polymorphism in leprosy patients with and without trophic ulcers: protocol for a case-control study [version 1; peer review: 1 approved with reservations]. F1000Research 2025, 14:57 (https://doi.org/10.12688/f1000research.157869.1)
NOTE: If applicable, it is important to ensure the information in square brackets after the title is included in all citations of this article.
track
receive updates on this article
Track an article to receive email alerts on any updates to this article.

Open Peer Review

Current Reviewer Status: ?
Key to Reviewer Statuses VIEW
ApprovedThe paper is scientifically sound in its current form and only minor, if any, improvements are suggested
Approved with reservations A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.
Not approvedFundamental flaws in the paper seriously undermine the findings and conclusions
Version 1
VERSION 1
PUBLISHED 09 Jan 2025
Views
10
Cite
Reviewer Report 29 Jul 2025
Khairuddin Djawad, dermatovenereologi, Hasanuddin University (Ringgold ID: 64739), Makassar, South Sulawesi, Indonesia;  Wahidin Sudirohusodo General Hospital, Makassar, Indonesia 
Approved with Reservations
VIEWS 10
This research has novel value regarding the polymorphism of the fokI gene in ulcers and non-ulcers in leprosy sufferers, but it is necessary to exclude possible confounding factors such as the possibility of other systemic diseases accompanying leprosy such as ... Continue reading
CITE
CITE
HOW TO CITE THIS REPORT
Djawad K. Reviewer Report For: Comparison of serum vitamin D level and vitamin D receptor gene FokI polymorphism in leprosy patients with and without trophic ulcers: protocol for a case-control study [version 1; peer review: 1 approved with reservations]. F1000Research 2025, 14:57 (https://doi.org/10.5256/f1000research.173385.r392598)
NOTE: it is important to ensure the information in square brackets after the title is included in all citations of this article.

Comments on this article Comments (0)

Version 1
VERSION 1 PUBLISHED 09 Jan 2025
Comment
Alongside their report, reviewers assign a status to the article:
Approved - the paper is scientifically sound in its current form and only minor, if any, improvements are suggested
Approved with reservations - A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.
Not approved - fundamental flaws in the paper seriously undermine the findings and conclusions
Sign In
If you've forgotten your password, please enter your email address below and we'll send you instructions on how to reset your password.

The email address should be the one you originally registered with F1000.

Email address not valid, please try again

You registered with F1000 via Google, so we cannot reset your password.

To sign in, please click here.

If you still need help with your Google account password, please click here.

You registered with F1000 via Facebook, so we cannot reset your password.

To sign in, please click here.

If you still need help with your Facebook account password, please click here.

Code not correct, please try again
Email us for further assistance.
Server error, please try again.