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

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.^9,11–13 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.^25–27 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.

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. Screening for excluded systemic conditions was conducted through structured clinical interviews and review of the patients’ medical records, including relevant laboratory and radiologic data when available. The control group will consist of leprosy patients without TU, who meet the same inclusion and exclusion criteria as the case group.

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. Anamnesis will include identity, sociodemographic information (e.g., education level, occupation, and household income), leprosy history (duration, treatment, and reaction history), sun exposure duration, smoking status, and ulcer history. Clinical data related to leprosy, including disease duration, WHO and Ridley-Jopling classification, treatment status, and history of reactions, will be obtained through patient interviews and verified from medical records where available. 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 was assessed based on clinical examination of the wound bed and further categorized using the Pressure Ulcer Scoring System (grades I to III). In cases where necrotic tissue or slough prevented adequate visualization, sharp debridement was performed as part of standard clinical care to facilitate accurate depth assessment. Only patients with ulcers confined to the subcutaneous layer were included, while those with wounds exposing deeper structures such as fascia, tendon, or bone were excluded. 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.

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:

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:

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

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 independently associated with the occurrence of TU in leprosy patients. In addition to vitamin D levels and FokI polymorphism, the model will consider relevant sociodemographic and clinical factors. Variables with a p-value < 0.25 in bivariate analysis will be considered for inclusion 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.

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.