Association between the rs1544410 polymorphism in the vitamin D receptor (VDR) gene and insulin secretion after gestational diabetes mellitus.

BACKGROUND AND AIMS: Genetic variants involved in vitamin D metabolism have been associated with diabetes and related syndromes/diseases. We wanted to investigate possible associations of polymorphisms in genes involved in vitamin D metabolism with indices of insulin resistance and insulin secretion, and also with development of diabetes after gestational diabetes mellitus (GDM).
MATERIALS AND METHODS: We have studied 376 women with previous GDM. Eight single nucleotide polymorphisms (SNPs) in the genes for vitamin D receptor (VDR) [rs731236, rs7975232, rs10735810, and rs1544410], vitamin D binding protein (DBP) [rs7041 and rs4588], and cytochrome P450 family 27 subfamily B member 1 (CYP27B1) [rs10877012 and rs4646536] were genotyped by TaqMan Allelic Discrimination Assay using the Quantstudio 7 Flex system. A 75-g oral glucose tolerance test (OGTT) was performed 1-2 years postpartum. The homeostasis model assessment of insulin resistance (HOMA-IR) and the disposition index [(insulinogenic index: I30/G30)/HOMA-IR] were used to calculate insulin resistance and insulin secretion, respectively. Serum samples for determination of 25(OH)D3 were collected at the time of the OGTT. Manifestation of diabetes was followed up to five years postpartum.
RESULTS: After adjustment for BMI, age, and ethnicity, the A-allele of the VDR rs1544410 polymorphism was found to be associated with increased disposition index (difference per allele = 3.56, 95% CI: 0.4567-6.674; p = 0.03). The A-allele of the DBP rs7041 polymorphism was found to be associated with 25(OH)D3 levels (difference [in nmol/L] per allele = -5.478, 95% CI: -8.315 to -2.641; p = 0.0002), as was the T-allele of the DBP rs4588 polymorphism (OR = -6.319, 95% CI: -9.466 to -3.171; p = 0.0001). None of the SNPs were significantly associated with HOMA-IR or postpartum diabetes.
CONCLUSIONS: This study provides evidence that the rs1544410 polymorphism of the VDR gene may be associated with increased insulin secretion in women after pregnancy complicated by GDM. Further studies in other populations are needed to confirm the results.

Introduction

Gestational diabetes mellitus (GDM) is defined as any degree of glucose intolerance with onset or first recognition during pregnancy [1]. The prevalence of GDM is increasing [2], in parallel with the increasing prevalence of type-2 diabetes (T2D) worldwide [3]. Women with a history of GDM have an increased risk of developing T2D relative to those with a normoglycemic pregnancy [4]. GDM is characterized by insulin resistance and beta-cell dysfunction, and the same is true for T2D [5].

Vitamin D has been implicated in several medical conditions [6]. It has been hypothesized that it may have a positive effect on insulin secretion and sensitivity. The mechanisms may be mediated by activation of the vitamin D receptor (VDR) on pancreatic beta cells and insulin-sensitive organs and also by regulation of calcium homeostasis [7, 8]. Studies have suggested that circulating concentrations of vitamin D may be inversely associated with the risk of diabetes, metabolic syndrome, insulin secretion, and insulin resistance [8-10]. Malik et al., in addition to a meta-analysis, have found associations between polymorphisms in the VDR gene and both diabetes and insulin resistance-related diseases [11, 12]. Moreover, single nucleotide polymorphisms (SNPs) in the vitamin D binding protein gene (DBP) affect 25(OH)D3 levels [13, 14] and increase the risk of T2D and the metabolic syndrome [15, 16]. There is also evidence of associations between polymorphisms in the CYP27B1 gene and type-1 diabetes [17, 18].

We have recently reported from the “Mamma Study” that vitamin D deficiency/insufficiency in women with a history of GDM appeared to be associated with beta-cell dysfunction and insulin resistance, but not with postpartum diabetes, after adjustment for factors that are well known to influence T2D [19]. We therefore hypothesized that 8 SNPs in genes known to be involved in the vitamin D metabolism, diabetes, and related traits (4 SNPs in VDR, 2 SNPs in DBP, and 2 SNPs in CYP27B1) [8-18] might be associated with insulin resistance and/or beta-cell function at 1–2 years postpartum and also development of diabetes after up to 5 years of follow-up in women with a history of GDM.

Material and methods

Patients

The design of the “Mamma Study” has been described in detail elsewhere [20]. Briefly, women delivering between 2003–2005 were invited to participate in the study, covering 86% of all pregnancies in the County of Skane in southern Sweden, including four of five delivery departments. The diagnosis of GDM was made using a 75-g oral glucose tolerance test (OGTT) at the twenty-eighth and/or the twelfth week of gestation. In the original evaluation, GDM was defined as two-hour capillary plasma glucose ≥10.0 mmol/l, gestational IGT as two-hour capillary plasma glucose 8.6–9.9 mmol/l, and normal glucose tolerance (NGT) during pregnancy as two-hour capillary plasma glucose <8.6 mmol/l. Among those who accepted to participate in the study and after exclusion of those who had already been diagnosed as having diabetes, 160 women had GDM, 309 had Gestational IGT and 167 had NGT. Women were followed for the development of diabetes using an OGTT at 1–2 years and 5 years after pregnancy, or until the diagnosis of diabetes. The World Health Organization (WHO) 1999 diagnostic criteria were used for classification of diabetes during follow-up [21]. Measurements of both glucose and insulin concentrations at 0, 30, and 120 min during OGTT at 1–2 years postpartum were performed to calculate indices of beta-cell function and insulin resistance, as previously reported [22]. Serum samples for determination of 25(OH)D3 were collected during the OGTT at 1–2 years postpartum. In the present investigation, we used the diagnostic criteria for GDM recommended by the WHO in 1999 [21]. Based on these criteria and on successful measurements of 25OHD3 concentrations, we identified 376 women who had previously had GDM and who formed the basis of the present study. All women gave their written informed consent and the Ethics Committee of Lund University approved the study (LU 259–00, 2002-04-17), which was performed in accordance with the Declaration of Helsinki.

Chemical analysis

Homeostasis model assessment was used to estimate insulin resistance [(HOMA-IR): (fasting serum insulin × fasting plasma glucose)/22.5] [23, 24]. Insulin secretory capacity was estimated using the insulinogenic index (I/G30): [insulin 30 min−insulin 0 min)/(glucose 30 min − glucose 0 min] [25]. As insulin resistance modulates insulin secretion, the disposition index was used to adjust insulin secretion for the degree of insulin resistance, obtained by dividing I/G30 by HOMA-IR [26]. The serum concentration of 25(OH)D3 (in nmol/L) was determined by liquid chromatography mass spectrophotometry (LC-MS/MS). Assays were performed according to accredited methods at the Department of Clinical Chemistry, Skåne University Hospital, Malmö, Sweden.

Genetic analysis

The SNPs were chosen for analysis based on previous associations with circulating vitamin D levels, diabetes, and related traits [8-18]. The following SNPs were genotyped: rs731236 (TaqI), rs7975232 (ApaI), rs10735810 (FokI), and rs1544410 (BsmI) in VDR; rs7041 and rs4588 in DBP; and rs10877012 and rs4646536 in CYP27B1. DNA was extracted from whole blood using the MaxiPrep Kit (QIAGEN, Sweden). SNPs were genotyped by TaqMan Allelic Discrimination Assay using the Quantstudio 7 Flex system. The genotyping results were confirmed by using positive controls during the genotyping as well as by re-genotyping of about 20% of the samples using the same genotyping method. The minor allele frequency for all SNPs was > 0.05. No deviation from Hardy-Weinberg equilibrium in the whole group or in the any of the subgroups was found. The success rate of genotyping was ≥96% (2 samples were not genotyped because of no available DNA). The genotyping protocol has been deposited in protocols.io. The DOI is: dx.doi.org/10.17504/protocols.io.bcjbiuin.

eQTL lookups

The association of the rs1544410 polymorphism with gene expression in human pancreatic islets was looked up in data from RNAseq data from 191 donors [https://www.biorxiv.org/content/10.1101/435743v2.full]. The data are uploaded with the following EGA accession numbers: RNAseq: EGAS00001004042, GWAS: EGAS00001004044 and Phenotype: EGAS00001004056.

Statistical analysis

All statistical analyses were performed using IBM SPSS Statistics for Windows, version 22.0 (IBM Corp., Armonk, NY). All genetic analyses were performed using PLINK version 1.07 (http://pngu.mgh.harvard.edu/~purcell/plink/index.shtml). Logistic and linear regressions with age, ethnicity, and body mass index (BMI) as covariates were used to estimate SNP associations and the data are presented as difference per allele or odds ratios (ORs) with 95% confidence intervals (CIs). The p-values are based on additive models for the genetic variants. Correction for multiple testing was performed using permutations.

Results

The clinical characteristics of the study subjects are presented in a previous publication [19]. The study included 376 women who were diagnosed with GDM during pregnancy in southern Sweden. At five years postpartum, 253 (67.3%) had normal glucose tolerance, 57 (15.2%) had diabetes, and for 66 (17.6%) there were missing data. The women were of European origin (n = 287, 76.3%) or non-European origin (n = 78, 20.8%) but 11 women (2.9%) with GDM were unclassifiable. The non-European group consisted of the subgroups Arab women (n = 33), Asian women (n = 35), and women of other origins (n = 10).

Of the SNPs tested, the A-allele of the rs1544410 polymorphism was associated with increased disposition index (difference per allele = 3.56, CI: 0.46–6.67; p = 0.03) using linear regression analysis with age, ethnicity, and BMI as covariates (Table 1).

Table 1

The association between the SNPs studied and disposition index.

SNP	Associated allele	Frequency of associated allele	Difference in disposition index per allele (95% CI)	p-value
rs7041	A	0.41	−0.47 (−2.71 to 1.76)	0.68
rs4588	T	0.38	−0.62 (−3.14 to 1.91)	0.63
rs731236	G	0.38	0.25 (−1.90 to 2.40)	0.82
rs7975232	C	0.40	−1.19 (−3.31 to 0.92)	0.27
rs10735810	A	0.38	0.62 (−1.59 to 2.82)	0.58
rs1544410	A	0.12	3.56 (0.46 to 6.67)	0.03
rs10877012	T	0.34	0.97 (−1.36 to 3.30)	0.42
rs4646536	G	0.34	1.05 (−1.30 to 3.37)	0.38

The associations were tested by using linear regression analysis with age, ethnicity, and BMI as covariates.

The rs731236 (Taql), rs7975232 (Apal), and rs1544410 (Bsml) polymorphisms of the VDR gene were in complete linkage disequilibrium (Fig 1), as previously shown [27]. However, the rs10735810 (FokI) polymorphism was not in linkage disequilibrium with the SNPs mentioned.

Fig 1

Linkage disequilibrium between the rs731236, rs7975232, rs10735810, and rs1544410 polymorphisms of the VDR gene.

However, we found no effect of the SNPs in question on the HOMA-IR, using linear multiple regression analysis (Table 2).

Table 2

The association between the SNPs studied and HOMA-IR.

SNP	Allele	Allele Frequency	Difference in HOMA-IR per allele (95% CI)	p-value
rs7041	A	0.41	0.05 (−0.14 to 0.24)	0.62
rs4588	T	0.38	0.01 (−0.20 to 0.22)	0.93
rs731236	G	0.38	−0.02 (−0.20 to 0.16)	0.83
rs7975232	C	0.40	0.01 (−0.16 to 0.18)	0.91
rs10735810	A	0.38	−0.07 (−0.26 to 0.12)	0.45
rs1544410	A	0.12	−0.07 (−0.34 to 0.20)	0.60
rs10877012	T	0.34	−0.05 (−0.25 to 0.15)	0.61
rs4646536	G	0.34	−0.004 (−0.20 to 0.20)	0.97

The associations were tested by using linear regression analysis with age, ethnicity, and BMI as covariates.

In logistic regression analysis with age, ethnicity, and BMI as covariates, the SNPs studied showed no associations with postpartum diabetes in women with a history of GDM (Table 3).

Table 3

The association between the SNPs studied and postpartum diabetes.

SNP	Associated allele	Frequency of associated allele	OR (95% CI)	p-value
rs7041	A	0.41	1.01 (0.66–1.54)	0.98
rs4588	G	0.62	1.42 (0.89–2.26)	0.14
rs731236	A	0.62	1.42 (0.95–2.12)	0.09
rs7975232	C	0.40	1.15 (0.77–1.72)	0.50
rs10735810	G	0.62	1.16 (0.76–1.78)	0.48
rs1544410	A	0.12	1.64 (0.81–3.30)	0.17
rs10877012	G	0.66	1.12 (0.73–1.73)	0.60
rs4646536	A	0.66	1.10 (0.71–1.68)	0.68

The associations were tested using logistic regression analysis with age, ethnicity, and BMI as covariates.

The A-allele of the rs7041 polymorphism of the DBP gene was associated with a reduction in circulating 25(OH)D3 (difference [in nmol/L] per allele = −5.48, 95% CI: −8.32 to −2.64; p = 0.0002). The same was observed for the T-allele of the rs4588 polymorphism (difference [in nmol/L] per allele = −6.32, 95% CI: −9.47 to −3.17; p = 0.0001). The other SNPs did not show any effect on circulating levels of 25(OH)D3 (Table 4).

Table 4

The association between the SNPs tested and circulating 25(OH)D3.

SNP	Associated allele	Frequency of associated allele	Difference in 25(OH)D3 (in nmol/L) per allele (95% CI)	p-value
rs7041	A	0.41	−5.48 (−8.32 to −2.64)	0.0002
rs4588	T	0.38	−6.32 (−9.47 to −3.17)	0.0001
rs731236	G	0.38	0.20 (−2.57 to −2.97)	0.89
rs7975232	C	0.40	1.01 (−1.71 to 3.73)	0.47
rs10735810	A	0.38	−0.87 (−3.78 to 2.05)	0.56
rs1544410	A	0.12	−0.66 (−4.75 to 3.42)	0.75
rs10877012	T	0.34	−1.16 (−4.14 to 1.82)	0.45
rs4646536	G	0.34	−1.14 (−4.08 to 1.81)	0.45

The associations were tested using linear regression analysis with age, ethnicity, and BMI as covariates

The rs1544410 polymorphism was an eQTL for the KAT8 regulatory NSL complex subunit 2 (KANSL2) and phosphofructokinase, muscle (PFKM) genes in human pancreatic islets [https://www.biorxiv.org/content/10.1101/435743v2.full] (Table 5). KANSL2 gene expression correlated with that of Islet Amyloid Polypeptide (IAPP) gene (Fig 2). PFKM expression correlated with stimulatory index (a measure of insulin secretion in islets), as well as with expression of insulin, glucagon, and IAPP genes (Fig 3) [https://www.biorxiv.org/content/10.1101/435743v2.full].

Table 5

The rs1544410 polymorphism was an eQTL for the KANSL2 and PFKM genes in human pancreatic islets.

SNP	SNP_rsid	Gene name	beta	t-stat	p-value
12:48239835	rs1544410	KANSL2	0,29472031	2,864151456	0,004661904
12:48239835	rs1544410	PFKM	0,223429837	2,167193122	0,031491255

“Lookup” of significant associations in human pancreatic islets from 191 donors

Fig 2

KANSL2 gene expression is correlated with that of IAPP gene.

Fig 3

The correlation of the PFKM expression with stimulatory index as well as with expression of insulin, glucagon, and IAPP genes.

Discussion

We have recently shown that vitamin D deficiency/insufficiency in women with a history of GDM is associated with beta-cell dysfunction and insulin resistance [19]. The key finding of the current study was that the A-allele of the rs1544410 polymorphism of the VDR gene was associated with increased insulin secretion, measured as disposition index in women with a history of GDM. VDR is a member of the nuclear receptor superfamily of transcriptional regulators. It forms a heterodimer with a retinoid X receptor (RXR) and is widely distributed throughout the tissues, including pancreatic beta cells [28]. The 25(OH)D3 is widely used in assessment of vitamin D status. 25(OH)D3 is converted in the liver to 1,25(OH)2D, which is the main ligand for VDR [29]. When 1,25(OH)2D binds to this complex, it leads to the transcription of several vitamin D-regulated genes [23]. A relatively recent study has shown increased expression and production of major renin-angiotensin system components in isolated islets from VDR knockout mice incubated under high-glucose conditions [30]. This was prevented and reversed by 1,25(OH)2D in parallel with an increase in glucose-stimulated insulin secretion [30]. Ogunkolade et al. have shown that the TaqI (rs731236) genotype of the VDR gene was an independent determinant of the insulin secretion index in healthy Bangladeshi adults, who have a high risk of developing T2D [27]. The FokI (rs10735810) polymorphism of the VDR gene was found to be an additional independent determinant of insulin secretion in individuals with vitamin D insufficiency [27]. Furthermore, VDR mRNA expression has been shown to be a determinant of insulin secretory capacity [27]. Unlike our study, there was no direct association of the BsmI (rs1544410) polymorphism with insulin secretion. Interestingly, the TaqI t allele (i.e. the G allele in the present study) and the BsmI b (G) allele may be susceptibility alleles for diabetes in the Kashmiri population [11]. This is consistent with our present finding that the opposite allele, the A-allele, of the rs1544410 polymorphism was associated with increased disposition index, and therefore would prevent the development of diabetes. On the other hand, the TaqI t homozygotes were found to be associated with the highest levels of insulin secretion index compared to TT and Tt genotypes in healthy Bangladeshi adults, who have a high risk of developing T2D [27]. However, it should be noted that there was no adjustment for the degree of insulin resistance (i.e. disposition index), as we have done in the present study. Another explanation might be that the susceptibility alleles differ in different populations. Previous studies have shown tight linkage disequilibrium between the ApaI, BsmI, and TaqI genotypes [27, 31], and the same was found in our study (Fig 1). Our finding that the rs1544410 polymorphism was an eQTL for the PFKM gene in human pancreatic islets (Table 4) and that the PFKM expression correlated with stimulatory index (a measure of insulin secretion in islets), as well as with expression of INS, GCG, and IAPP genes (Fig 3) supports the role of this SNP in the insulin secretory capacity.

In the Rancho Bernardo study, the BsmI bb (GG) genotype was found to be associated with HOMA-IR after adjustment for age, sex, BMI, calcium, and vitamin D use in older adults without diabetes [32]. In the present study, we found no association between the polymorphisms tested and the HOMA-IR. Interestingly, a recent meta-analysis provided evidence of an association between VDR BsmI polymorphism and metabolic syndrome, supporting the idea that the VDR BsmI variant G allele may be a susceptibility marker of metabolic syndrome [12]. On the other hand, gene carriers with the VDR BsmI BB (AA) genotype have been found to have significantly higher levels of fasting glucose than gene carriers with the genotype Bb or bb in young men from Germany with low physical activity, but the effect was absent in men with a high degree of physical activity [33]. This discrepancy in the risk allele perhaps represents a gender difference.

In addition, we found that the A-allele of the rs7041 polymorphism and the T-allele of the rs4588 polymorphism of the DBP gene were associated with lower levels of circulating 25(OH)D3. The composite genotype of these 2 SNPs (rs7041 and rs4588) results in different DBP isotypes (Gc1f, Gc1s, and Gc2). These isotypes are known to influence the binding affinity of DBP and circulating vitamin D levels [34, 35]. Our results are consistent with previous findings that T-allele carriers (i.e. A-allele carriers in this study) of the rs7041 polymorphism had less circulating 25(OH)D3, regardless of reproductive state among third-trimester pregnant, lactating, and non-pregnant/non-lactating women [13]. Moreover, the TT genotype of the rs7041 polymorphism was found to be associated with metabolic syndrome in PCOS women, and with significantly lower 25(OH)D3 levels in both PCOS and control groups [15]. This was also shown in Jordanian subjects, where the genotypes containing the variant allele of rs7041 (TT, TG) and rs4588 (AA, AC) (i.e. T-allele in this study) were found to be associated with lower 25(OH)D3 levels than the wild-type genotypes (GG and CC, respectively) [14]. Interestingly, Rahman et al. found that the Glu/Glu genotype of the rs7041 and the Lys/Lys genotype of the rs4588 variants of DBP gene were significantly higher in subjects with type 2 diabetes than controls [16].

We have not observed any association between the SNPs studied and postpartum diabetes in the present study, which could be due to the small number of women who developed diabetes, to differences in ethnic background, to differences in study design, or simply to absence of an effect of these SNPs on the development of postpartum diabetes.

The main strength of the study was that we used the disposition index derived from the OGTT as a measure of beta-cell function, adjusted for insulin resistance. This is a more accurate measure of insulin secretory capacity than the HOMA-b [25]. However, the study still lacked enough statistical power (i.e. > 80%) to detect small potential effects of the SNPs in question on prediction of postpartum diabetes.

In conclusion, our study provides evidence that the rs1544410 polymorphism of the VDR gene may be associated with insulin secretion. Further studies in other populations will be needed to confirm the results.

(SAV)

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2 Jan 2020

PONE-D-19-32028

Association between the rs1544410 polymorphism in the vitamin D receptor ​(VDR) gene and insulin secretion after gestational diabetes mellitus

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Reviewer #1: The study aimed to investigate possible associations of polymorphisms in genes involved in vitamin D metabolism with indices of insulin resistance and insulin secretion, and also with development of diabetes after gestational diabetes mellitus (GDM), the manuscript was found interesting, The subject selection and how subjects move through the study is clear. It is a good work.

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I request the authors to clear the following queries

1. Explain how the study size was arrived at.

Authors’ reply: Briefly, women delivering between 2003–2005 were invited to participate in the study, covering 86% of all pregnancies in the County of Skane in southern Sweden, including four of five delivery departments. The diagnosis of GDM was made using a 75-g oral glucose tolerance test (OGTT) at the twenty-eighth and/or the twelfth week of gestation. In the original evaluation, GDM was defined as two-hour capillary plasma glucose ≥10.0 mmol/l, gestational IGT as two-hour capillary plasma glucose 8.6–9.9 mmol/l, and normal glucose tolerance (NGT) during pregnancy as two-hour capillary plasma glucose <8.6 mmol/l. Among those who accepted to participate in the study and after exclusion of those who had already been diagnosed as having diabetes, 160 women had GDM, 309 had Gestational IGT and 167 had NGT. Women were followed for the development of diabetes using an OGTT at 1–2 years and 5 years after pregnancy, or until the diagnosis of diabetes. The World Health Organization (WHO) 1999 diagnostic criteria were used for classification of diabetes during follow-up. Measurements of both glucose and insulin concentrations at 0, 30, and 120 min during OGTT at 1‒2 years postpartum were performed to calculate indices of beta-cell function and insulin resistance, as previously reported. Serum samples for determination of 25(OH)D3 were collected during the OGTT at 1‒2 years postpartum. In the present investigation, we used the diagnostic criteria for GDM recommended by the WHO in 1999. Based on these criteria and on successful measurements of 25OHD3 concentrations, we identified 376 women who had previously had GDM and who formed the basis of the present study.

We have now revised the Section “Material and methods/Patients” accordingly.

2. Did the authors confirmed the genotyping results using any other methods (sequencing etc.)

Authors’ reply: As the TaqMan Allelic Discrimination Assay is well validated method for SNP genotyping, we have not used any other methods to confirm the genotyping. However, we used positive controls during the genotyping. Moreover, the genotyping results were confirmed by re-genotyping of about 20% of the samples using the same genotyping method.

We think this would be enough to confirm our results and hope it is accepted according to your journal policies. We have now added this information to the Section “Material and methods/Genetic analysis”.

3. Please include the genotyping raw data (spss file etc.) as supplementary information.

Authors’ reply: We would like to bring to your attention that we discovered that two samples of the studied samples were not genotyped because of no available DNA. We have now added this information to the Section “Material and methods/Genetic analysis”. The genotyping raw data is included in SPSS file as supplementary information. Please, let us to know if you need more raw data (t. ex. original genotyping raw data from the TaqMan ® Genotyper ™ Software).

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Authors’ reply: No changes to our financial disclosure have been made.

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Authors’ reply: The genotyping protocol have been deposited in protocols.io. The DOI is: dx.doi.org/10.17504/protocols.io.bcjbiuin

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Authors’ reply: The data are uploaded. The EGA accession numbers are:

RNAseq: EGAS00001004042

GWAS: EGAS00001004044

Phenotype: EGAS00001004056

This information has been added to the revised manuscript.

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Authors’ reply: We have now provided data within the manuscript. Please, see “Table 2” in the Results Section.

4. Thank you for stating the following in the Acknowledgments Section of your manuscript:

"The study was supported by grants from the Research Funds of Skåne University Hospital and

289 the Skåne County Council Research and Development Foundation. The funders had no role in

290 study design, data collection and analysis, decision to publish, or preparation of the manuscript.".

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Authors’ reply: We have removed any funding-related text from the manuscript. We want to update our funding information as follow: “The study was supported by grants from the Research Funds of Skåne University Hospital, the Skåne County Council Research and Development Foundation and ALF Region Skåne. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.”

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Reviewer #1: Yes

Reviewer #2: Yes

Authors’ reply: There are no questions raised by the reviewer.

________________________________________

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

Reviewer #1: Yes

Reviewer #2: Yes

Authors’ reply: There are no questions raised by the reviewer.

________________________________________

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Reviewer #2: Yes

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________________________________________

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Reviewer #1: The study aimed to investigate possible associations of polymorphisms in genes involved in vitamin D metabolism with indices of insulin resistance and insulin secretion, and also with development of diabetes after gestational diabetes mellitus (GDM), the manuscript was found interesting, The subject selection and how subjects move through the study is clear. It is a good work.

Authors’ reply: There are no questions raised by the reviewer.

Reviewer #2: I would like to appreciate the work done by the authors submitted for publication. Overall the manuscript is well designed, experimental study is well executed, statistical data analysis performed is acceptable. Overall the manuscript is well written and the subject of study and findings reported are relevant.

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________________________________________

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Reviewer #1: Yes: Moushira Erfan Zaki

Reviewer #2: No

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Submitted filename: Response to Reviewers.doc

Click here for additional data file.

13 Apr 2020

Association between the rs1544410 polymorphism in the vitamin D receptor ​(VDR) gene and insulin secretion after gestational diabetes mellitus

PONE-D-19-32028R1

Dear Dr. Shaat,

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

Within one week, you will receive an e-mail containing information on the amendments required prior to publication. When all required modifications have been addressed, you will receive a formal acceptance letter and your manuscript will proceed to our production department and be scheduled for publication.

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With kind regards,

Narasimha Reddy Parine, Ph.D

Academic Editor

PLOS ONE

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 #1: All comments have been addressed

Reviewer #2: 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 #1: Yes

Reviewer #2: Yes

**********

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

Reviewer #1: Yes

Reviewer #2: 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 #1: Yes

Reviewer #2: 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 #1: Yes

Reviewer #2: 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 #1: no comments for authors, good work, the conclusion showed that he overall early ANC booking in Ethiopia was very low compared with

WHO recommendation for pregnant women. Both sociodemographic and obstetric

factors significantly affect early initiation of ANC. Strategies targeting advocating

women education and standard piece of information concerning what pregnant women

should expect and do during the prenatal period, universal access of family planning

and prenatal care service and establishment village outreach health facility should be

addressed by Ministry of Health and its stake holders to improve early initiation of ANC.

Reviewer #2: (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.

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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: Moushira Zaki, National Research Centre,Egypt

Reviewer #2: Yes: Prof. Dr. Nadeem Sheikh

7 May 2020

PONE-D-19-32028R1

Association between the rs1544410 polymorphism in the vitamin D receptor ​(VDR) gene and insulin secretion after gestational diabetes mellitus

Dear Dr. Shaat:

I am 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 notify them about your upcoming paper at this point, to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, 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.

For any other questions or concerns, please email plosone@plos.org.

Thank you for submitting your work to PLOS ONE.

With kind regards,

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on behalf of

Dr. Narasimha Reddy Parine

Academic Editor

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