Genetic variations of CYP2R1 (rs10741657) in Bangladeshi adults with low serum 25(OH)D level-A pilot study.

BACKGROUND: Some studies revealed that despite having sufficient sun exposure and dietary supply, the level of serum 25(OH)D in Bangladeshi adults is lower than its normal range. Genetic pattern of an individual is also an essential factor that regulates the level of serum 25(OH)D. However, the genetic variations of CYP2R1 (rs10741657) and their association with low serum 25(OH)D level in Bangladeshi adults are yet to be explored.
OBJECTIVE: This study was conducted to determine the frequency of variants of rs10741657 of CYP2R1 gene and its association with low serum 25(OH)D level among Bangladeshi adults.
METHOD: This pilot study was conducted among thirty individuals with low serum 25(OH)D level as the study population and ten subjects with sufficient serum 25(OH)D level as controls based on the inclusion and exclusion criteria. Genetic analysis of rs10741657 of CYP2R1 including primer designing, DNA extraction, PCR of target region with purification and Sanger sequencing of the PCR products were done accordingly. For statistical analysis, One-way ANOVA followed by LSD test, Freeman-Halton extension of Fisher's exact test, Chi-square test (χ2) test and unpaired student t-test were performed.
RESULTS: In this study, genetic variants of CYP2R1 (rs10741657) among the study population were genotype GG (63.30%), GA (30%) and AA (6.7%). Minor allele frequency of the study population was 0.217. The association between GG and GA genotypes of CYP2R1 (rs10741657) with low serum 25(OH)D level among the study population was found and it was statistically significant. Statistically significant differences were also observed between the genotypes and alleles of the study population and controls.
CONCLUSIONS: The presence of 'GG' and 'GA' genotypes of rs1041657 in CYP2R1 gene is associated with low serum 25(OH)D level among Bangladeshi adults in this pilot study.

Introduction

In human physiology vitamin D is appreciated as a very important nutrient since its identification. The structure of vitamin D3 was first determined in 1936 and its activation process was established in 1971 [1]. Over the last few decades, it has been proven that vitamin D is an essential nutrient for a number of physiological functions [1, 2]. Inadequacy of vitamin D level in human body is associated with rickets and osteomalacia as well as a wide range of other illnesses like various cancers and several autoimmune diseases, psychological and cardiovascular disorders. It has been observed that about 1 billion people have vitamin D deficiency or insufficiency all over the world [3]. Vitamin D deficiency is a condition when the serum 25(OH)D level is below 20 ng/ml and vitamin D insufficiency is when the serum 25(OH)D level is 21–29 ng/ml [4]. Both these conditions are termed as low serum 25(OH)D level [5]. According to various studies around 51–100% of the different groups of population suffered from low vitamin D level [3, 6–8]. In spite of adequate sun exposure, a recent study showed that about 70.7% people living in coastal area of Cox’s Bazar, Bangladesh had low serum 25(OH)D level [9].

Sunlight (UVB exposure), seasonal variation, skin color, age, race, gender, BMI and dietary/supplemental vitamin D intake are some of the major factors that influence serum vitamin D level of an individual [10]. Genetic pattern is also one of the key factors that may cause variable serum vitamin D level [11]. In recent times, a number of molecular conceptions regarding vitamin D level have been added to genome-wide association studies [12]. It has been revealed that genetic factors may affect the bioavailability of vitamin D in the circulation of human being ranging from 53% to 68.9% [13]. The presence or absence of various single nucleotide polymorphisms (SNPs) in vitamin D related genes is one such factor. The common vitamin D related genes are–CYP2R1, DHCR7, DBP, VDR, CYP24A1 and CYP27B1. Among these, CYP2R1 (cytochrome P450 family 2 subfamily R polypeptide 1) encodes for 25-hydroxylase enzyme. This enzyme is responsible for the formation of 25(OH)D in liver [13, 14].

It has been observed that variants of rs10741657 in CYP2R1 gene may give rise to low serum 25(OH)D level irrespective of adequate sun exposure and healthy diet [15]. Despite abundant sun exposure low serum 25(OH)D level has been found among most Bangladeshi people [8, 9]. The genetic influence of rs10741657 of CYP2R1 on low serum 25(OH)D level has never been investigated before in Bangladesh.

Hence, the goal of the study was to assess the frequency of the genetic variants of CYP2R1 (rs10741657) in Bangladeshi adults with low serum 25(OH)D level and also in Bangladeshi adults with sufficient serum 25(OH)D level. Additionally, possible associations between the genotypes of CYP2R1 (rs10741657) and serum 25(OH)D level of the adults with low serum 25(OH)D level and also with those of controls were investigated.

Materials and methods

Study design

This cross-sectional study was carried out after obtaining the ethical clearance from the Research Review Committee of the Department of Physiology and Ethical Review Committee of Dhaka Medical College, Dhaka, Bangladesh from July 2019 to June 2020. Permission was taken from respective authorities of the Center for Medical Biotechnology, MIS (Management Information System), DGHS (Directorate General of Health Service), Dhaka, Bangladesh, for the genetic analysis and Dhaka Metropolitan Police, clubs of Abahani field, Dhaka and Good neighbors Bangladesh, an NGO, for collecting the samples (S1 Appendix). To conduct this study 18–60 years old Bangladeshi middle class (assessed by Kuppuswamy socio-economic scale [16]) males and females (non- veiled) with light brown complexion (assessed by Fitzpatrick scale), normal BMI (18.5–24.9 kg/m2) and sun exposure of at least 45 minutes between 11 am to 2 pm were selected. Umbrella or sun block users, smokers, pregnant or lactating mothers and people with pathological conditions like chronic liver disease, chronic renal disease, malabsorption syndrome, chronic pulmonary disease; systemic disease like Diabetes mellitus were excluded from this study. People taking vitamin D or calcium supplements, estrogen or progesterone replacement therapy or medications such as steroid (5mg/day for last 3 months), anticonvulsant, thiazide diuretics, antifungal agents were also excluded. Those exclusions were done based on their history of chronic diseases and estimation of their serum creatinine, glutamic pyruvic transaminase level, prothrombin time and random blood glucose. Serum calcium and albumin level were also estimated to exclude any pathological condition related to serum calcium and albumin level. All the study subjects were healthy individuals with no other medical problems. Finally, Traffic Police, Community NGO workers (Health Workers), Cricket and Football players were included as the study subjects. Prior to the collection of their blood samples, all participants were informed about the study process and the written consent were taken.

Study subjects

Since it was a pilot study the subject numbers were kept at 30 adults with low serum 25(OH)D level as study population and 10 controls with sufficient serum 25(OH)D level for the analysis of rs10741657 of CYP2R1 gene. The level of Serum 25(OH)D <20 ng/ml, 21–29.9 ng/ml and ≥30 ng/ml were considered as serum 25(OH)D level deficiency, insufficiency and sufficiency accordingly and both insufficiency and deficiency were considered as low serum 25(OH)D level for the current study. Blood sample collection was done until the accomplishment of the sample size. As a result, a total of 54 blood samples were collected in the beginning of the study. Based upon the investigation reports of serum 25(OH)D and other parameters, 2 subjects were excluded for having higher serum creatinine level than normal. Among the rest, 30 subjects were found with low serum 25(OH)D level (<30 ng/ml) whereas the other 22 were found with sufficient serum 25(OH)D level (≥30 ng/ml). Finally, 30 subjects with low serum 25(OH)D level were selected as the study population and 10 subjects with sufficient serum 25(OH)D level were selected as the controls for comparison. The controls were selected from those 22 samples with sufficient serum 25(OH)D level using a random table of Microsoft excel 2010 software.

Biochemical analysis

Serum 25(OH)D levels of the participants were estimated by chemiluminescence immunoassay using MAGLUMI series Fully- auto chemiluminescence immunoassay analyzer (intra-assay CV 4.99%, inter-assay CV 5.93%). Serum calcium, creatinine, albumin and SGPT were measured in Automated Biochemistry Analyzer. Measurement of random blood glucose was done by Hexokinase method in Automated Biochemistry Analyzer and Prothrombin time (PT) was analyzed on automated coagulation analyzer. All those biochemical analyses were performed at the department of pathology, Dhaka medical college, Dhaka, Bangladesh.

Molecular analysis

For genetic analysis of rs10741657 of CYP2R1 the blood samples were carried to Center for Medical Biotechnology, MIS, DGHS, Dhaka, Bangladesh and stored at -70ºC freezer. Genomic DNA was extracted and isolated from the samples using Promega reliaPrepTM Blood gDNA isolation kit (Promega, USA). After taken out from the freezer, the frozen blood samples thawed completely at room temperature. Proteinase K enzyme was used to break the cellular proteins of the samples. Cell lysis buffer was added after that to breakdown the cells and the DNA came out in this process. Then the samples were kept at 56ºC in a heat block machine for 10 minutes. Binding buffer was mixed to bind with the DNA of the samples and column wash solution was applied to wash away the waste thereafter. Finally, the DNA of the samples were stored with nucleus free water at -20ºC freezer. The information of selected SNP rs10741657 was taken from NCBI database. The website, “https://wheat.pw.usda.gov/demos/BatchPrimer3/” was used for primer designing. Then it was validated by ‘IDT OligoAnalyzer Tool’ of Integrated DNA Technologists. The polymerase chain reaction (PCR) of rs10741657 was performed using forward and reverse primers:

5’-TTCAATAATCAGAAGCAAACAAAAAGTGC-3’

and 5’-GGTTTTAAGCCATCAGATTGGTGGTAAT-3’.

Polymerase Chain Reaction (PCR) of extracted DNA was done using GoTag G2 Green PCR master mix (Thermofisher) and the designed primers of rs10741657. The whole procedure was performed under a biological safety cabinet to prevent DNA contamination. Several attempts were taken to achieve a good PCR product. The volume, temperatures, PCR cycles and DNA amount for a good PCR product were optimized. Finally, Polymerase Chain Reaction of a volume of 30 μl solution was done consisted of 15 μl PCR master mix, 1.5μl(50ng) extracted genomic DNA, 075 μl forward primer, 0.75 μl reverse primer and 12 μl nucleus free water. PCR reaction profile was carried out by initial denaturation at 95°C for 3 min, followed by 32 cycles of denaturation at 95°C for 30 sec, annealing at 58.04°C for 30 sec and a final extension at 72°C for 5 min. After that, Gel electrophoresis was done to check the PCR product whether the desired PCR product was present or not. Then all the PCR products were purified by QIAquick PCR Product Purification Kit (QIAGEN PCR Product Purification Kit). The Sequencing of the purified PCR products was performed by ABI Sanger Sequencer 3500 device. The ABI-prism Bigdye terminator version 3.1 sequencing ready reaction kit was used in cycle sequencing. For data analysis, the AB1 files generated by Sanger Sequencer were imported to the Chromas software using Chromas® Software version 2.66. The chromatogram data was then edited and aligned to find out the good quality data and saved as SCF file. For interpretation of the mutation data, query sequence was compared with available known BLAST database and the SNP has been identified. Then the saved SCF files were converted to FASTA format for the use in Mega 7 software. All the final sequencing data of the study subjects were uploaded and published on NCBI GenBank (S2 Appendix).

Statistical analysis

Allele and Gene frequencies with a percentage of rs10741657 were analyzed among the study population (N = 30) and the controls (Nc = 10). Statistical analysis to see the association between low serum 25(OH)D level and the genotypes of CYP2R1 (rs10741657) in the study population was performed by One way ANOVA followed by LSD test. Freeman-Halton extension of Fisher Exact Test was done to compare between the genotypes, whereas Chi-squared Test was done to compare between the alleles of the study population and controls. Unpaired student t-test was performed to compare between sociodemographic and biochemical variables of the study population and controls. A difference was considered as statistically significant if p value was <0.05. Statistical analysis was done by using a computer based statistical program SPSS version 25.0.

Result

The socio-demographic characteristics and biochemical parameters of the study population and controls are shown in Tables 1 and 2, respectively. Among the study population and controls, all other parameters were within the normal ranges except serum 25(OH)D level (Table 2).

Table 1

Socio-demographic characteristics of the study subjects (Nt = 40).

Parameters	Study subjects (N = 30)	Controls (Nc = 10)	P value
Age (Years)	34.60±12.87	28.90±9.26	0.205 ns
Sex
Male	25 (83.30%)	07 (70%)	0.374 ns
Female	05 (16.70%)	03 (30%)
BMI (kg/m2)	22.59±1.13	22.23±1.02	0.374 ns
SBP (mmHg)	116.33 ± 7.18	114.50 ± 4.37	0.45 ns
DBP (mmHg)	75.50 ± 6.34	77.00 ± 5.86	0.5136 ns
Occupation
Traffic Police	14(46.70%)	02(20%)	0.066 ns
Player	11(36.70%)	05(50%)
NGO worker	05(16.70%)	03(30%)

Results were expressed as mean ±SD, frequency and percentage; % = Percent; BMI = body mass index; SBP = systolic blood pressure; DBP = diastolic blood pressure; N = total number of study population; Nc = total number of controls; Figure in parenthesis shows percentage. Blood pressure was measured manually by stethoscope and sphygmomanometer during data collection; Unpaired student t-test was performed to compare between two groups. The test of significance was calculated for all comparisons and p value <0.05 was accepted as level of significance.

ns = not significant,

* = significant.

Table 2

Biochemical parameters of the study subjects (Nt = 40).

Parameters	Study subjects (N = 30)	Controls (Nc = 10)	P value
Serum 25(OH)D (ng/ml)	25.22±2.15	49.64±15.25	0.0001 *
Serum Albumin (gm/dl)	4.34±0.40	4.06±0.30	0.05 ns
Fasting blood glucose (mmol/L)	4.49±0.51	4.84±0.36	0.05 ns
SGPT (U/L)	17.53±5.56	22.60±6.09	0.02*
Serum Creatinine (mg/dl)	1.048±0.18	0.99±0.174	0.461 ns
Prothrombin time (second)	12.60±0.85	13.20±1.54	0.017*
Serum Calcium (mg/dl)	9.10±0.39	9.51±0.40	0.007*

Results were expressed as mean and standard deviation (mean ± SD); 25(OH)D = 25-hydroxy vitamin D; SGPT = serum glutamic pyruvic transaminase; N = total number of study population; Nc = total number of controls. Unpaired student t-test was performed to compare between two groups. The test of significance was calculated for all comparisons and p value <0.05 was accepted as level of significance.

ns = not significant,

* = significant.

Genotypes of rs107416567 of CYP2R1 gene

The genotyping data of rs10741657 of CYP2R1 of the study subjects were derived from Sanger sequencing (Figs 1–3). There were 3 variants of genotype in rs10741657 of CYP2R1 gene: genotype GG, GA and AA.

Fig 1

Sample of chromatogram data of rs10741657 showing G allele in location 26 bp.

Fig 3

Sample of chromatogram data of rs10741657 showing the SNP- A allele in location 14 bp.

Among the study population the mean difference of serum 25(OH)D level of the variant genotype groups (GG, GA and AA) was statistically significant. The mean serum 25(OH)D level of genotype group GG was lower than group GA and group AA. The serum 25(OH)D level was also found to be lower in genotype group GA than group AA. The mean difference of serum 25(OH)D level among the genotype group GG and GA was statistically significant. So, it can be emphasized that the genotype GG and GA of rs10741657 were associated with low serum 25(OH)D level among the study population and there was no association of any of the genotypes of rs10741657 with sufficient serum 25(OH)D level among the controls (Table 3). There was a statistically significant difference between the variant genotypes of rs10741657 of CYP2R1 gene of the study population and the controls (p = 0.033) (Table 4). In addition, it was also found that in the study population the distribution of allele G was 78.3% whereas of allele A was 21.7%. While in the control group, the distribution of allele G was 45% and that of allele A was 55%. This showed a statistically significant difference between the alleles of rs10741657 of CYP2R1 gene in the study population and the controls (p = 0.010).

Table 3

Serum 25(OH)D level among the genotypes of rs10741657 in CYP2R1 gene of the study population (N = 30) and Controls (Nc = 10).

Serum 25(OH)D level among the genotypes of rs10741657 among the study population
Genotype (n) vs Genotype (n)	Serum 25(OH)D level (ng/ml)	p-value
GG (19) vs GA (09) vs AA (02)	24.46±1.18 vs 26.51±2.22 vs 26.63±1.80	0.033*
GG (19) vs GA (09)	24.46±1.18 vs 26.51±2.22	0.016*
GA (09) vs AA (02)	26.51±2.22 vs 26.63±1.80	0.937 ns
GG (19) vs AA (02)	24.46±1.18 vs 26.63±1.80	0.149 ns
Serum 25(OH)D level among the genotypes of rs10741657 among the controls
Genotype (n) vs Genotype (n)	Serum 25(OH)D level (ng/ml)	p-value
GG (02) vs GA (05) vs AA (03)	37.49±2.48vs 48.60±15.82 vs 59.46±15.68	0.314ns
GG (02) vs GA (05)	37.49±2.48 vs 48.60±15.82	0.395ns
GA (05) vs AA (03)	48.60±15.82 vs 59.46±15.68	0.344ns
GG (19) vs AA (02)	37.49±2.48 vs 59.46±15.68	0.145ns

Results were expressed as Mean ±SD. One way ANOVA followed by LSD test was performed to compare between groups; N = Total number of study population, nGG = number of genotype GG, nGA = number of genotype GA, nAA = number of genotype AA; The test of significance was calculated for all comparisons and p value <0.05 was accepted as level of significance.

ns = not significant,

* = significant.

Table 4

Comparison of serum 25(OH)D level and genotypes of the study subjects (Nt = 40).

Serum 25(OH)D Level	Genotypes of rs10741657 in CYP2R1 gene			p-value
	GG (ntGG = 21)	GA (ntGA = 14)	AA (ntAA = 5)
Subjects with low serum 25(OH)D (N = 30)	19 (63.3%)	09 (30%)	02 (6.7%)	0.033*
Controls with sufficient serum 25(OH)D (Nc = 10)	02 (20%)	05 (50%)	03 (30%)

Freeman-Halton extension of Fisher’s exact test was done to analyze data; Nt = Total number of subjects; N = Total number of study population; Nc = Total number of controls; ntGG = Total number of genotype GG in study subjects; ntGA = Total number of genotype GA in study subjects; ntAA = Total number of genotype AA in study subjects; Figure in parenthesis shows percentage; The test of significance was calculated for all comparisons and p value <0.05 was accepted as level of significance,

* = significant.

Discussion

The present pilot study revealed that GG, GA, and AA are the variants of CYP2R1 (rs10741657) in Bangladeshi adults. Similar variants of rs10741657 in CYP2R1 gene were found in studies from different countries like Saudi Arabia, Singapore, Jordan, and other countries [10, 13, 15, 17, 18]. The rs10741657 of CYP2R1 gene was found to be a predictor of serum 25(OH)D level in several other studies [19, 20]. This rs10741657 was associated with low level of serum 25(OH)D in Caucasian subjects [21]. Peterson et al. [22], had performed a longitudinal study in Denmark where they found the children carrying genotype GG and GA of rs10741657 in CYP2R1 were associated with low serum 25(OH)D level during the Winter and Spring, whereas it was interestingly observed that children with genotype AA always had the higher mean serum 25(OH)D level in all three seasonal cycles. In addition, Zhang et al.’s [11] study also identified GG genotype in rs10741657 to be associated with low serum 25(OH)D level. These findings were consistent with our study as well, where we found that the maximum number of the people in our study population who had low serum 25(OH)D level were with the genotypes GG and GA. On the contrary, in a study which was conducted amongst Korean population, it was found that there was no association of serum 25(OH)D level with any variants of rs10741657 in CYP2R1 gene [23]. Thereby this lends us to think that there could be some role of natural selection in the process amongst different ethnicity.

rs10741657 is an important site located at the promoter region of CYP2R1 gene which encodes 25-hydroxylase enzyme. Variation of rs10741657 can affect the rate and activity of this enzyme. Since it was found in the current study that the percentage of study population having genotypes GG and GA to be higher than those with genotype AA, so there might be a possibility that the presence of “G allele” instead of “A allele” in rs10741657 plays a role in reducing the activity or rate of the production of 25-hydroxylase enzyme. In one study a group of healthy Caucasian subjects with allele G were found to have low serum 25(OH)D level despite six months of artificial UVB therapy with a slight increase from their baseline [24]. Similarly, another study also found that allele G (GG and GA variants) of rs10741657 had minimum response whereas serum 25(OH)D level increased by 2.5 times in genotype AA after 9 weeks of vitamin D supplementation among Iranian adolescent girls [25]. So, it can be hypothesized that the presence of G allele (Genotype GG and GA) of rs10741657 may have a role in the presence of low level of serum 25(OH)D among individuals. However, the mechanistic aspect of allele G’s influence on 25-hydroxylase enzyme and subsequent serum level of 25(OH)D is still not understood clearly.

Based on the ethnicity of this study, the minor allele of rs10741657 in CYP2R1 gene of the study population was A and the minor allele frequency was 0.217. This was different in Jordanian (0.35), Caucasian (0.42), Lebanese (0.29), Virginian (0.42), Arabian (0.33) and South Asian (0.36) people as per different other studies [15, 17, 18]. According to the Genbank data of NCBI, the higher percentages of A allele (minor allele) were found among the group of people living in northern latitudes like- Northern Sweden (0.48), Estonia (0.42), Korea (0.40), UK (0.39) compared to the tropical and subtropical countries [26]. So, there might be a possibility of natural selection where people from the tropical and sub-tropical region carry higher percentages of allele G and lower percentages of allele A compared to the people from the countries with shorter duration of daylight.

Our results could potentially be explained further by the aspect of the geographical location of Bangladesh. Bangladesh is located at the latitude of 20°34’ to 26°38’ North and 88°01’ to 92°41’ East longitude. The sun exposure is sufficient and people living here are getting adequate sunlight [27]. In this present study the study population and control group both received adequate sun exposure on daily basis and on their socio-economic status was homogenous and other biochemical parameters were within normal range. It was also observed that the frequency of GG was more frequent than the frequency AA among the study population, which could be an instance where this variation of the genetic makeup of the CYP2R1 (rs10741657) among Bangladeshi adults is influenced by the process of natural selection. The study population had values within normal range for serum calcium level along with some other biochemical markers (SGPT, PT, FBS, Serum Creatinine) and those parameters were also normal in the control group. As the study population had low serum 25(OH)D level despite the above-mentioned biochemical markers being in normal range and they did not exhibit any history of low Vitamin D related disorders, it can be assumed that the low serum 25(OH)D level of study population could naturally be adequate to perform their physiological functions.

There could be a recommendation to carry out similar study with a larger sample size while observing other biochemical markers (like Parathyroid hormone), as well across other tropical and sub-tropical countries including Bangladesh to assess the influence of other vitamin D related gene variations to reach a more definitive conclusion. Thereby it would be advisable to do a review of the reference values of serum 25(OH)D level used in medical science for the assessment of serum vitamin D level and tailor it to different ethnicity and geographies accordingly. Correspondingly, it could be advisable to conduct the routine investigation for polymorphisms of Vitamin D related genes among individuals. The implication of this study in medical science could be that the people with polymorphic vitamin D related genes may have different reference values for their physiological assessments.

Limitation

Genetic analysis of other reference sequencing numbers of various genes which might be associated with low serum 25(OH)D level of CYP2R1 gene could not be done due to time and financial constraints. Moreover, it was a pilot study in Bangladesh and we could not include a large number of study population.

Conclusion

This pilot study has demonstrated that the presence of ‘GG’ and ‘GA’ genotypes of rs10741657 of CYP2R1 are associated with low serum 25(OH)D level among Bangladeshi adults. From this study we can anticipate that the requirement of serum 25(OH)D level could vary from individual to individual to perform their physiologic functions. Since the study sample size was small and other vitamin D related gene variations could not be considered, it remains to be elucidated further through a more robust and expansive study including different regions of Bangladesh to definitively conclude and work towards considering to redefine reference values of serum 25(OH)D level for Bangladeshi adults in the field of medical science for more tailored approach for the diagnosis and treatment purpose in Bangladesh.

Ethical committee and corresponding permissions.

(DOCX)

Click here for additional data file.

NCBI extension numbers of sequence files.

(DOCX)

Click here for additional data file.

Available data of the study subjects.

(DOCX)

Click here for additional data file.

20 Sep 2021

PONE-D-21-25624Genetic Variations of CYP2R1 (rs10741657) in Bangladeshi adults with low serum 25(OH)D levelPLOS ONE

Dear Dr. Rahman,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process. Besides the important reviewers' comments, authors should address the following points:

Please, follow appropriate reporting guidelines, such as STROBE

Please, better specify the objectives of the study; it would be better to add the word to determine “frequency” of variants…)

The small sample size is a major limitation. Authors stated that this is a pilot study (this should be better also included in the title, discussion, and conclusion).

Regarding Data availability, authors stated “Yes - all data are fully available without restriction” and data are “within the manuscript and its as supporting information files”. However, the submission doesn’t contain unidentified patients’ data. According to PLOS ONE publication criteria, authors are required to make all data underlying the findings described fully available, without restriction, and from the time of publication. PLOS allows rare exceptions to address legal and ethical concerns, that should be clearly explained.

Authors should clearly describe participants’ inclusion and exclusion criteria, time of recruitment, how participants were selected (convenience sample?)

Authors should provide definitions/diagnostic criteria for described conditions/outcomes, including vitamin D deficiency.

The authors didn’t study other genetic determinants (other vitamin D associated genes), which could be possible confounders.

In statistical analysis, were data tested for normality (some data are reported as mean (SD) but appear not normally distributed). What about confounders?

Study results and tables should be reported in the results section.

Tables require better organization with comparison of variables between the study and control groups.

In figure 1, it would be better to annotate the polymorphisms.

Figures 2 & 3 seem to be of little value as its data can be incorporated in tables or text.

Discussion requires major revision, critically discussing main findings of study (without repeating results) and study implications.

In light of being a pilot, the current conclusion seems to overestimate study findings; authors should reduce the strength of their statement, confirming that this is a pilot study, and recommend further well-powered studies.

Please, revise the references to follow PLOS ONE style.

Authors should revise the manuscript for several errors in language, sentence structure, grammar, and punctuation.

Please submit your revised manuscript by Nov 04 2021 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.

A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.

An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.

We look forward to receiving your revised manuscript.

Kind regards,

Elsayed Abdelkreem, MD, PhD

Academic Editor

PLOS ONE

Journal Requirements:

When submitting your revision, we need you to address these additional requirements.

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at

https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and

https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

2. Please ensure that you refer to Figure 3 in your text as, if accepted, production will need this reference to link the reader to the figure.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Partly

Reviewer #2: Yes

Reviewer #3: Yes

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: I Don't Know

Reviewer #2: Yes

Reviewer #3: I Don't Know

**********

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: No

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: I have a major concern regarding the sample size of the study. With the current small sample size and the used study design, the study probably is underpowered. Was the sample size and power of the study was calculated prior starting the study?

There are other points to consider as follows:

1. In line 73 (in the introduction), Please remove DBP as GC is already mentioned and It is the gene encoding vitamin D binding protein (DBP). In addition, please include CYP27B1 gene with the other major common vitamin D genes as it is one of the significant genes involved in vitamin D metabolism (it is the gene encoding 1α-hydroxylase, the enzyme responsible for the 2nd step of activation of vitamin D in the kidney).

2. Please mention the exclusion and inclusion criteria in the methods and the inter and intra-assay CV for the performed tests.

3. Please move Table 1 and 2 to the Results section.

4. Information in line 186-187 (in the results) is already mentioned in the methods, please remove.

Reviewer #2: First of all, I would like to congratulate the researchers for this very good work. The "CONCLUSION" section is also finished with a very good conclusion. However, the deficiency that I see in the study and other similar studies is that it is in the form of statistically researching the negativities associated with vitamin D deficiency and making a connection between them. This should provide an advantage if a selective mutation is dominant in geographic regions where this mutation is present. What advantage does vitamin d provide at lower levels? What protection does it provide for people in those areas? The answer to this question should also be explored in the comments. Natural selection never happens without a reason. When I look at it from the point of view of the country I live in, kidney stones come to mind. But I think this should be investigated. If researchers can add an answer and comment to this question in terms of their own country, I think that their work will be more beneficial than other studies. Beautiful original work worth publishing. The journal meets all publication criteria. I wish the researchers success in their new work.

Reviewer #3: Thank you for your work. Some comments

1. Grammar and sentence construction i nthe entire manuscript have to be revised/ improved

2. What is the significance of yorus tidu if this has been reported in the literature already?

3.inclusion and exclusion criteria not stated

4. how di you come up with the final nuber of subjects and how did you choose the initial of 54. please state clearly how you got yoru subjects

5. what was the significantce of all the other biochemical parameters that were taken apart form vitamin D levels

6. for tables 1 and 2 make them reader friendly-remove too many lines and make it cleaner. yo ulaso did not specify the statistical significance betwenn the two groups of all the parameters

7. for table 3 you did not compare the results between groups. you only compared them within the groyp. it wuld be ebtter if you compared for exampe the genoype GG of affected and controls and the corresponding statistical significance

8. i nthe discussion, do not repeat anymore your results but instead interpret and explain them. your tables hsould be self explanatory and in the discussion you will only give its importance

9. what is the impact of the study?

10. what is the implciation i nterms of treatment?

11. make the discussion mroe concise

**********

6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: Yes: Shatha Alharazy

Reviewer #2: No

Reviewer #3: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

29 Sep 2021

Responses to academic editor

• Please, follow appropriate reporting guidelines, such as STROBE

Response: Thank you for your kind concern. In this 1st revised version of the manuscript, we have tried to maintain it accordingly.

• Please, better specify the objectives of the study; it would be better to add the word to determine “frequency” of variants…)

Response: This has been done in the current version of the manuscript. Please check page no. 2, line no. 33 and page no. 2, line no. 84 to 88.

• The small sample size is a major limitation. Authors stated that this is a pilot study (this should be better also included in the title, discussion, and conclusion).

Response: Thank you so much, it was really needed. This has been done in the current version of the manuscript. Please check page no. 1, line no. 5, and page no. 13, line no. 258 and page no. 16, line no. 326.

Regarding Data availability, authors stated “Yes - all data are fully available without restriction” and data are “within the manuscript and its as supporting information files”. However, the submission doesn’t contain unidentified patients’ data. According to PLOS ONE publication criteria, authors are required to make all data underlying the findings described fully available, without restriction, and from the time of publication. PLOS allows rare exceptions to address legal and ethical concerns, that should be clearly explained.

Response: All the data are now uploaded in a new file, but the details of the study subjects are hidden to maintain their privacy. All the gene data with NCBI links are already given in the previous submission.

• Authors should clearly describe participants’ inclusion and exclusion criteria, time of recruitment, how participants were selected (convenience sample?)

Response: This has been done in the current version of the manuscript. Please check page no. 4, line no. 92 to page no. 5, line no. 112.

• Authors should provide definitions/diagnostic criteria for described conditions/outcomes, including vitamin D deficiency.

Response: This has been done in the current version of the manuscript. Please check page no. 3, line no. 58 to 60 and page no. 6, line 118 to 122.

• The authors didn’t study other genetic determinants (other vitamin D associated genes), which could be possible confounders.

Response: This was the limitation of the study. As it was a part of an academic research project, we could not analyze other genetic determinants due to time and financial constraints. In addition, this has been explained in the current version of the manuscript. Please check page no.16, line no. 318 to 323 and page no.17, line no. 338 to 341.

• In statistical analysis, were data tested for normality (some data are reported as mean (SD) but appear not normally distributed). What about confounders?

Response: Yes, all data were tested for normality by the normal Q- Q plot of Shapiro- Wilk Normality Test (SPSS version 23 software). Mean (SD) of age, BMI, FBS, serum Ca++, creatinine, SGPT and albumin level of the study subjects and serum 25(OH)D level of the study population were apparently normally distributed. But serum 25(OH)D level of the controls were not normally distributed. The confounders could not be clearly understood for the mean serum 25(OH)D level of the controls as the number of the controls was only 10 and other genetic determinants could not be analyzed due to financial constraints.

• Study results and tables should be reported in the results section.

Response: This has been done in the current version of the manuscript. Please check page no. 9, line no. 195 and page no. 10, line no. 206.

• Tables require better organization with comparison of variables between the study and control groups.

Response: This has been done in the current version of the manuscript. Please check page no. 9, line no. 195, page no. 10, line no. 206 and page no. 13, line no. 245.

• In figure 1, it would be better to annotate the polymorphisms.

Response: This has been done in the current version of the manuscript.

• Figures 2 & 3 seem to be of little value as its data can be incorporated in tables or text.

Response: Figures 2 & 3 have been replaced by new figures and the previous data are incorporated in text in the current version.

• Discussion requires major revision, critically discussing main findings of study (without repeating results) and study implications.

Response: This has been done in the current version of the manuscript.

• In light of being a pilot, the current conclusion seems to overestimate study findings; authors should reduce the strength of their statement, confirming that this is a pilot study, and recommend further well-powered studies.

Response: Those have been changed in the current version. Please check page no. 16, line no. 326 to page no. 17, line no. 334.

• Please, revise the references to follow PLOS ONE style.

Response: This has been done in the current version.

• Authors should revise the manuscript for several errors in language, sentence structure, grammar, and punctuation.

Response: We have tried our best to follow proper language, sentence structure, grammar and punctuation in the current manuscript.

Responses to Reviewer#1

• I have a major concern regarding the sample size of the study. With the current small sample size and the used study design, the study probably is underpowered. Was the sample size and power of the study was calculated prior starting the study?

Response: Thank you for your kind concern. This was a pilot study and none of the vitamin D influencing gene has ever been analyzed in Bangladesh. Moreover, as it was an academic research project, only 30 subjects as study population with low serum 25(OH)D level and 10 subjects sufficient serum 25(OH)D level as controls were included due to time and financial constraints. Therefore, we could not calculate the sample size and power of the study prior to the research.

• In line 73 (in the introduction), Please remove DBP as GC is already mentioned and It is the gene encoding vitamin D binding protein (DBP). In addition, please include CYP27B1 gene with the other major common vitamin D genes as it is one of the significant genes involved in vitamin D metabolism (it is the gene encoding 1α-hydroxylase, the enzyme responsible for the 2nd step of activation of D in the kidney).

Response: This has been done in the current version of the manuscript. Please check page no. 4, line no. 74.

• Please mention the exclusion and inclusion criteria in the methods and the inter and intra-assay CV for the performed tests.

Response: This has been done in the current version of the manuscript. Please check page no. 5 line no. 98 to 112 and page no. 6, line no. 131 and 132. Except serum 25(OH)D level, the intra and inter assay CV of other parameters were not estimated. Other samples were analyzed for a single time as there was a scarcity of laboratory reagents due to financial constraints. This is because serum 25(OH)D level and genetic variants of CYP2R1 (rs10741657) were our study parameter and other parameters were estimated to confirm only the exclusion and inclusion criteria.

• Please move Table 1 and 2 to the Results section.

Response: This has been done in the current version of the manuscript. Please check page no. 9, line no. 195 and page no. 10, line no. 206.

• Information in line 186-187 (in the results) is already mentioned in the methods, please remove.

Response: This has been removed in the current version of the manuscript.

Responses to Reviewer#2

• First of all, I would like to congratulate the researchers for this very good work. The "CONCLUSION" section is also finished with a very good conclusion. However, the deficiency that I see in the study and other similar studies is that it is in the form of statistically researching the negativities associated with vitamin D deficiency and making a connection between them. This should provide an advantage if a selective mutation is dominant in geographic regions where this mutation is present. What advantage does vitamin d provide at lower levels? What protection does it provide for people in those areas? The answer to this question should also be explored in the comments. Natural selection never happens without a reason. When I look at it from the point of view of the country I live in, kidney stones come to mind. But I think this should be investigated. If researchers can add an answer and comment to this question in terms of their own country, I think that their work will be more beneficial than other studies. Beautiful original work worth publishing. The journal meets all publication criteria. I wish the researchers success in their new work.

Response: Thank you so much for your kind consideration. Actually, Bangladesh is a developing country and execution of such molecular research projects of any level is quite challenging. You may understand by the fact that this is the first genetic analysis which was carried out in Dhaka Medical College, Bangladesh. As, it was an academic study, it was to complete the whole research work in time considering the recent pandemic. Hence, your kind notes of appreciation have encouraged us to continue in this field of research in future.

The aforementioned concerns have been included in the current version of the revised manuscript. Please check page no. 15 to 17.

Responses to Reviewer#3

• Grammar and sentence construction i nthe entire manuscript have to be revised/ improved

Response: Thank you for your kind concern. This has been revised in the current version.

• What is the significance of yorus tidu if this has been reported in the literature already?

Response: This study has never been conducted in Bangladesh. As a pilot study, our goal was to determine the variants of CYP2R1 (rs10741657) gene and their association with low serum vitamin D level among Bangladeshi adults. The objectives have been elaborated in the current version, please check page no. 4, line no. 80 to 88.

• inclusion and exclusion criteria not stated

Response: This has been stated in the current version of the manuscript. Please check page no. 5, line no. 98 to page no.6, line no 122.

• how di you come up with the final nuber of subjects and how did you choose the initial of 54. please state clearly how you got yoru subjects

Response: Thank you so much again to give us a chance to clear about the fact. This was a pilot study and none of the vitamin D influencing gene has ever been analyzed in Bangladesh. Moreover, as it was an academic research project, only 30 subjects as study population with low serum 25(OH)D level and 10 subjects sufficient serum 25(OH)D level as controls were included due to time and financial constraints. This has been explained in the current version, please check page no. 6, line no. 122 to 136, and page no. 5, line no. 98 to 114.

• what was the significantce of all the other biochemical parameters that were taken apart form vitamin D levels

Response: This has been explained in the current version, please check page no. 5, line no. 107 to 111.

• for tables 1 and 2 make them reader friendly-remove too many lines and make it cleaner. yo ulaso did not specify the statistical significance betwenn the two groups of all the parameters

Response: This has been done in the current version of the manuscript. Please check page no. 9, line no. 195 and page no. 10, line no. 206.

• for table 3 you did not compare the results between groups. you only compared them within the groyp. it wuld be ebtter if you compared for exampe the genoype GG of affected and controls and the corresponding statistical significance

Response: Thank you for your concern, in table 3, only 2 controls had genotype GG, so the comparison between the groups was not done. In addition, the sample size was small and we took only 10 controls to see if there was any variation among the genotypes and alleles between study population and controls. For your kind concern we have added a new table to explain it. This has been explained in page no. 13, line no. 245 to 254 (Table 4).

• i nthe discussion, do not repeat anymore your results but instead interpret and explain them. your tables hsould be self explanatory and in the discussion you will only give its importance

Response: Thank you so much for your kind advice. This has been corrected in the current version.

• what is the impact of the study?

Response: This has been explained in the current version. Please check page no. 16, line no. 313 to page no. 17, line no. 334.

• what is the implciation i nterms of treatment?

Response: This has been explained in the current version. Please check page no. 16, line no. 318 to page no. 17, line no. 334.

• make the discussion mroe concise

Response: This has been corrected in the current version accordingly. Please check page no. line no.

Thank you very much for considering our manuscript for revision.

Yours Sincerely,

Kazi Lutfar Rahman

Email: klrahman@yahoo.com

Submitted filename: Response to Reviewers.docx

Click here for additional data file.

12 Oct 2021

PONE-D-21-25624R1Genetic Variations of CYP2R1 (rs10741657) in Bangladeshi adults with low serum 25(OH)D level- A Pilot StudyPLOS ONE

Dear Dr. Rahman,

Thank you for submitting your manuscript to PLOS ONE. The revised manuscript is much better. However, some minor, but important, comments have to be addressed.   You are kindly requested to respond to some concerns raised by Reviewer #3. Moreover, authors should briefly emphasize, in the results section, most important findings in the table (not just saying data are shown in tables). Last, the limitations should be moved to appear before the conclusion (could be integrated with the last paragraph in the discussion)

Please submit your revised manuscript by Nov 26 2021 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.

A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.

An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.

We look forward to receiving your revised manuscript.

Kind regards,

Elsayed Abdelkreem, MD, PhD

Academic Editor

PLOS ONE

Journal Requirements:

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #2: All comments have been addressed

Reviewer #3: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #2: Yes

Reviewer #3: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #2: Yes

Reviewer #3: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #2: (No Response)

Reviewer #3: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #2: (No Response)

Reviewer #3: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #2: Since I think that this and similar studies, which have been done despite limited technical possibilities, should be supported, I would like to be approved for publication. Good luck to the researchers.

Reviewer #3: Thank you for your revisions. The manuscript has improved a lot i nterms of grammar, sentence construction, tables, and explanation of results

I still have some minor comments though. (I would like to apologize for all the typo errors during my initial comments).

1. You did not discuss the relevance of the significant differences in the biochemical parameters like sgpt, calcium, PT between subjects and controls. What were the reference values for these parameters? Or they were both in the normal ranges for both groups thats why they were not discussed anymore?

2. Clarification: were your subjects all healthy with incidental findings of low vitamin D levels? pls state that they were all healthy "meaning no other medical problem?"

3. I also wanted to know what is the implication in terms of management. Will you recommend vitamin D supplement to those who have low levels? If you will not give supplement, again what is the significance of the study if you will not be able to translate it to clinical practice? Likewise, did you consider checking if the subjects had concomittant disorders associated with low vitamin D levels? In your introduction you said that low vitamin D is related to several disorders. How can you correlate it with your results-- did you ask the medical histories of your subjects and check if these could be related to low vitamin D? (or are they all healthy?)

4. Will you also recommend to test for this polymorphism routinely among the population? If yes, what is the significance. Will this be harmful for their health?

5. For a better organization, I suggest created subheadings in your methodology. First discuss about the "study subjects" then the " biochemical analysis" and lastly "molecular analysis". Do not lump them altogether.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #2: No

Reviewer #3: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

24 Oct 2021

Responses to academic editor

• You are kindly requested to respond to some concerns raised by Reviewer #3. Moreover, authors should briefly emphasize, in the results section, most important findings in the table (not just saying data are shown in tables). Last, the limitations should be moved to appear before the conclusion (could be integrated with the last paragraph in the discussion)

Response: Thank you so much for your appreciation of the revised version. All the concerns raised by Reviewer #3 are responded accordingly in this current version of the manuscript. Most important findings of the table are emphasized in the current version, please check page no. 09, line no. 194 to 195 and page no. 11, line no. 232 to page no. 12, line no. 245. The limitations section is moved to appear before the conclusion as you had suggested, please check page no. 17, line no. 344 to 349.

According to the journal requirement, all the references have been revised carefully. In the first round of revision of the manuscript, irrelevant references were removed and only relevant references were kept and added. In this round of revision, we have carefully checked that no changes and retractions have been made to the references. Currently, the references which have been used all are available and not retracted.

In the first revised version, we removed and added some of our references which were not mistakenly elaborated in previous rebuttal letter and now those are added here,

Reference:

Zgheib, Nathalie & Arabi, Asma & Mahfouz, Rami & El-Hajj Fuleihan, Ghada. (2013). OC002—Cyp2r1 Genetic Polymorphisms Are Associated With Lower 25-Hydroxy Vitamin D Levels In Lebanese Subjects. Clinical Therapeutics. 35. e1. 10.1016/j.clinthera.2013.07.003.

Status: Removed

Comment: The information for which it was that had been modified and the modified information is available in no.15 article of the current version.

Reference:

Holick MF, Binkley NC, Bischoff-Ferrari HA, Gordon CM, Hanley DA, Heaney RP, et al. Evaluation, treatment, and prevention of vitamin D deficiency: An endocrine society clinical practice guideline. J Clin Endocrinol Metab. 2011;96: 1911–1930. doi:10.1210/jc.2011-0385

Status: added

Comment: It was used to define the refence value of serum 25(OH)D level, (ref no.4 in current version)

Reference:

Binkley N, Ramamurthy R, Krueger D. Low vitamin D status: Definition, prevalence, consequences, and correction. Endocrinol Metab Clin North Am. 2010;39: 287–301. doi:10.1016/j.ecl.2010.02.008

Status: added

Commenet: It was used to define the low serum 25(OH)D level, (ref no.5 in current version)

Reference:

Ahmed B, Shiraji KH, Chowdhury MHK, Uddin MG, Islam SN, Hossain S. Socio-economic Status of the Patients with Acute Coronary Syndrome: Data from a District-level General Hospital of Bangladesh. Cardiovasc J. 2017;10: 17–20. doi:10.3329/cardio.v10i1.34357

Status: added

Comment: It was used to determine the socioeconomic status of the study subjects, (ref no.16 in current version)

Reference:

Islam MA, Alam MS, Sharker KK, Nandi SK. Estimation of Solar Radiation on Horizontal and Tilted Surface over Bangladesh. Comput Water, Energy, Environ Eng. 2016;05: 54–69. doi:10.4236/cweee.2016.52006

Status: added

Comment:

It was used to determine sun exposure according to geographic location of Bangladesh (ref no.27 in current version)

Finally, the reference numbers were rearranged according to the article rearrangement during revision.

Responses to Reviewer#3

• You did not discuss the relevance of the significant differences in the biochemical parameters like sgpt, calcium, PT between subjects and controls. What were the reference values for these parameters? Or they were both in the normal ranges for both groups that’s why they were not discussed anymore?

Response: Thank you so much for your concern to make a better version of this article. All the biochemical parameters were within the normal ranges among both the study population and the controls, so they were not discussed anymore. Here, we have added the chart that we followed to ensure the normal range of all the biochemical parameters.

Reference values of different parameters

Parameters Normal range

1. Serum calcium 8.50-10.30 mg/dl (Automate

Biochemistry Analyzer)

2. Serum albumin 3.40 – 5.00 gm/dl (Automated Biochemistry Analyzer)

3. Fasting blood glucose 3.30 – 6.11 mmol/L (Automated Biochemistry

Analyzer)

4. Serum Creatinine Male: 0.60 1.50

Female: 0.40 1.20

Child: 0.20 0.70 (Automated Biochemistry Analyzer)

5. Serum SGPT < 40 U/L (Automated Biochemistry Analyzer)

6. Serum 25(OH)D Deficiency:<20ng/ml

Insufficiency: 20-29.9ng/ml

Sufficiency: ≥30ng/ml

7. Prothrombin time 11-16 second.

• Clarification: were your subjects all healthy with incidental finding of low vitamin D levels? pls state that they were all healthy “meaning no medical problem?”

Response: Thank you for your concern. Yes, all the subjects were healthy and based on your suggestion we have added this clarification in the methodology. Please check page no.5, line no.111.

• I also wanted to know what is the implication in terms of management. Will you recommend vitamin D supplement to those who have low levels? If you will not give supplement, again what is the significance of the study if you will not be able to translate it to clinical practice? Likewise, did you consider checking if the subjects had concomitant disorders associated with low serum vitamin D level? In your introduction you said that low vitamin D is related to several disorders. How can you correlate it with your result- did you ask the medical histories of your subjects and check if these could be related to low vitamin D? (or are they all healthy?)

Response: Thank you for your concern. We began the process by taking the medical history of the study subjects. Their medical history did not reveal any pathological conditions related to low serum 25(OH)D level. In addition to this, we investigated serum calcium level to determine any abnormalities associated with calcium homeostasis. We had a financial constraint for which we were not able to assess serum PTH and other investigations which might be associated with any vitamin D related abnormalities. Since it was a pilot study, the sample size was small. For better results, we need to perform this study on a larger scale. In this pilot study, we have observed that people with low serum 25(OH)D level can perform their physiological functions without any hardship and it is evidenced by the investigation results and also by the history of having no vitamin D related disorders. As all the study subjects were apparently healthy individuals, so it can be hypothesized that due to natural selection, which could be based on the geographical location and sun exposure, variants of rs10741657 (GG, GA) can perform their physiological functions with low serum 25(OH)D level without any need for any kind of vitamin D supplements. But again, to come to a better conclusion, we need further studies with larger sample size and other vitamin D related biochemical analysis. The major implication of this study is to consider the genetic influence in order to redefine the cutoff value of low serum vitamin D level. All these are explained elaborately in the discussion and the conclusion portion of the manuscript, please check page no.16, 17 and 18.

• Will you also recommend to test for this polymorphism routinely among the population? If yes, what is the significance. Will this be harmful for their health?

Response: Since it was a pilot study with a small sample size, we need to further assess to confirm the genetic influence of this polymorphism on vitamin D level on a larger scale. Only then we can recommend if this polymorphism can routinely be done among the population or not. If the precautions are properly taken, it will not be harmful for the health. It has been added in the current version please check page no.17, line no.339 to 342.

• For a better organization, I suggest created subheadings in your methodology. First discuss about the ‘Study Subjects’ the “biochemical analysis” and lastly “molecular analysis”. Do not lump them altogether.

Response: Thank you for your concern, these have been done in the current version. Please check page no. 5 line no. 116, page no. 6 line no. 132 and page no. 7 line no. 141.

Submitted filename: Response to Reviewers.docx

Click here for additional data file.

8 Nov 2021

Genetic Variations of CYP2R1 (rs10741657) in Bangladeshi adults with low serum 25(OH)D level- A Pilot Study

PONE-D-21-25624R2

Dear Dr. Rahman,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Elsayed Abdelkreem, MD, PhD

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #3: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #3: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #3: I Don't Know

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #3: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #3: No

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #3: (No Response)

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #3: No

10 Nov 2021

PONE-D-21-25624R2

Genetic Variations of CYP2R1 (rs10741657) in Bangladeshi adults with low serum 25(OH)D level -A Pilot Study

Dear Dr. Rahman:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

If we can help with anything else, please email us at plosone@plos.org.

Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Elsayed Abdelkreem

Academic Editor

PLOS ONE