Neeraj Tomar

Prevalence of vitamin D deficiency and its relationship with thyroid autoimmunity in Asian Indians: a community-based survey.

25-Hydroxy vitamin D (25(OH)D) deficiency is linked with predisposition to autoimmune type 1 diabetes and multiple sclerosis. Our objective was to assess the relationship between serum 25(OH)D levels and thyroid autoimmunity. Subjects included students, teachers and staff aged 16-60 years (total 642, 244 males, 398 females). Serum free thyroxine, thyroid-stimulating hormone (TSH), and thyroid peroxidase autoantibodies (TPOAb), intact parathyroid hormone and 25(OH)D were measured by electrochemiluminescence and RIA, respectively. Thyroid dysfunction was defined if (1) serum TSH > or = 5 microU/ml and TPOAb>34 IU/ml or (2) TSH > or = 10 microU/ml but normal TPOAb. The mean serum 25(OH)D of the study subjects was 17.5 (sd 10.2) nmol/l with 87 % having values < or = 25 nmol/l. TPOAb positivity was observed in 21 % of subjects. The relationship between 25(OH)D and TPOAb was assessed with and without controlling for age and showed significant inverse correlation (r - 0.08, P = 0.04) when adjusted for age. The prevalence of TPOAb and thyroid dysfunction were comparable between subjects stratified according to serum 25(OH)D into two groups either at cut-off of < or = 25 or >25 nmol/l or first and second tertiles. Serum 25(OH)D values show only weak inverse correlation with TPOAb titres. The presence of such weak association and narrow range of serum 25(OH)D did not allow us to interpret the present results in terms of quantitative cut-off values of serum 25(OH)D. Further studies in vitamin D-sufficient populations with wider range of serum 25(OH)D levels are required to substantiate the findings of the current study.

Pattern of 25-hydroxy vitamin D response at short (2 month) and long (1 year) interval after 8 weeks of oral supplementation with cholecalciferol in Asian Indians with chronic hypovitaminosis D

Hypovitaminosis D is common in Asian Indians. Physicians often prescribe 1500 mug (60 000 IU) cholecalciferol per week for 8 weeks for vitamin D deficiency in India. Its efficacy to increase serum 25-hydroxy vitamin D (25(OH)D) over short (2 months) and long (1 year) term is not known. We supplemented a group of twenty-eight apparently healthy Asian Indians detected to have low serum 25(OH)D (mean 13.5 (sd 3.0) nmol/l) on screening during January-March 2005. Serum parathyroid hormone (PTH) level was supranormal in 30 % of them. Oral supplementation included 1500 mug cholecalciferol per week and 1g elemental Ca daily for 8 weeks. Serum 25(OH)D, total Ca, inorganic P and intact (i) PTH were reassessed in twenty-three subjects (twelve females and eleven males) who had follow up at both 8 weeks and 1 year. At 8 weeks the mean 25(OH)D levels increased to 82.4 (sd 20.7) nmol/l and serum PTH normalized in all. Twenty-two of the twenty-three subjects had 25(OH)D levels>49.9 nmol/l. At 1 year, though the mean 25(OH)D level of 24.7 (sd 10.9) nmol/l was significantly higher than the baseline, all subjects were 25(OH)D deficient. Five subjects with supranormal iPTH at baseline showed recurrence of biochemical hyperparathyroidism. Thus, with 8 weeks of cholecalciferol supplementation in Asian Indians with chronic hypovitaminosis D, mean serum 25(OH)D levels would be normalized and serum PTH value would be reduced to half. However, such quick supplementation would not maintain their 25(OH)D levels in the sufficient range for 1 year. For sustained improvement in 25(OH)D levels vitamin D supplementation has to be ongoing after the initial cholecalciferol loading.

Calcium-Sensing Receptor Autoantibodies and Idiopathic Hypoparathyroidism

CONTEXT Data on calcium-sensing receptor autoantibodies (CaSRAbs) in hypoparathyroidism are variable. OBJECTIVE We assessed the prevalence and significance of CaSRAbs in idiopathic hypoparathyroidism. DESIGN This was a case-control study. SUBJECTS One hundred forty-seven patients with idiopathic hypoparathyroidism treated during 1998-2011 in a tertiary care setting and 348 controls [healthy, n = 199; type 1 diabetes mellitus (T1DM), n = 99; and chronic lymphocytic thyroiditis (CLT), n = 50] participated in the study. METHODS CaSRAb assays included Western blot with CaSR protein expressed in Escherichia coli or human embryonic kidney (HEK)-293 cells, immunoprecipitation (IP) using in vitro-transcribed/translated protein, and indirect immunofluorescence on HEK293-CaSR. Functional significance was assessed by ERK1/2 phosphorylation. PTH and CaSR genes were sequenced for mutations. RESULTS E coli-Western blot assay revealed 16.3% CaSRAb positivity in idiopathic hypoparathyroidism, which was comparable with healthy subjects and CLT but significantly less than the T1DM controls. The prevalence of CaSRAbs on HEK293-Western blot (24.5%) against 150 kDa and/or 168 kDa protein in hypoparathyroidism was significantly higher than the healthy subjects, T1DM, and CLT. IP assay showed CaSRAbs in 12.9% of the hypoparathyroid patients but not in controls. The sensitivity and specificity of CaSRAbs in E coli and HEK-293-CaSR Western blot and IP assays were 16.3% and 83.1%, 24.5% and 88.9%, and 12.9% and 100%, respectively, and 42.1% of the cases detected were common in the IP assay and HEK293-Western blot. Duration of illness and coexistent autoimmunity were similar in patients with and without CaSRAbs. The CaSRAb-positive sera showed no immunofluorescence and phosphorylated ERK1/2 activity. The CaSR gene sequence was normal in all patients. One of the patients showed a novel p.Met1_Asp6del mutation in the signal peptide region of the PTH gene. CONCLUSION IP performed the best in detecting CaSRAbs in 12.9% of hypoparathyroid patients. Although CaSRAbs were functionally inert, its clinical relevance remains due to 100% specificity. Limited prevalence of CaSRAb suggests heterogeneity in the etiology of idiopathic hypoparathyroidism or the presence of CaSR epitopes other than those measured in the current study.

Prevalence and Significance of NALP5 Autoantibodies in Patients with Idiopathic Hypoparathyroidism

CONTEXT Role of parathyroid autoimmunity in idiopathic hypoparathyroidism (IH) is not clear. Recently, parathyroid-specific NACHT leucine-rich-repeat protein 5 (NALP5) autoantibodies (Ab) have been reported in autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy syndrome. OBJECTIVE Our objective was to assess prevalence and significance of NALP5 Ab in patients with IH. DESIGN AND SETTING This was a case-control study at a tertiary care hospital. SUBJECTS Subjects included 145 patients with IH recruited from 1998-2011 and 152 healthy controls. METHODS Immunoprecipitation (IP) and Western blot (WB) assays were performed using ³⁵S-labeled NALP5 protein produced by in vitro transcription-translation and recombinant NALP5 protein in Escherichia coli, respectively. AIRE gene sequencing was performed in NALP5 Ab-positive patients. RESULTS One of 145 patients (0.69%) and none of the 152 controls had NALP5 Ab on IP assay. Nine of 147 patients (6.12%) and four of 152 controls (2.63%) had NALP5 Ab on WB. One patient with NALP5 Ab on IP (36.6 sd score), also positive on WB, had a frameshift p.Ala386Serfs*38 AIRE gene mutation and adrenocortical Ab. Eight subjects with NALP5 Ab detected on WB had normal AIRE gene sequence. CONCLUSIONS IP is currently the best assay to detect clinically relevant NALP5 Ab. Presence of NALP5 Ab in only one patient with IH who also had AIRE gene mutation suggests that these Ab are exceptionally rare in IH (0.69%) and, when present, occur in context of the autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy syndrome.

Presence of spondyloarthropathy and its clinical profile in patients with hypoparathyroidism.

Background  Though spondyloarthropathy has been described in patients with sporadic idiopathic hypoparathyroidism (SIH), the clinical profile is not known.

Effect of vitamin D supplementation on cathelicidin, IFN-γ, IL-4 and Th1/Th2 transcription factors in young healthy females.

Objectives:We assessed the effect of cholecalciferol and calcium supplementation on mRNA expression of cathelicidin (LL-37), Th1 and Th2 cytokines and their transcription factors in the peripheral blood mononuclear cells (PBMCs) in healthy females with vitamin D deficiency (VDD).Subjects/Methods:Subjects included 131 females with biochemical VDD randomized to receive (a) oral cholecalciferol (60 000 IU/week for 8 weeks followed by 60 000 IU/fortnight (b) calcium (elemental calcium 500 mg twice/day) (c), dual supplementation and (d) placebo for 6 months. The mRNA expression of cathelicidin, Th1 (IFN-γ) and Th2 (IL-4 and its antagonist-IL-4δ2) cytokines and their transcription factors (T-bet, STAT4, GATA-3, STAT6) were measured in the PBMC by real-time PCR before and after intervention.Results:Cholecalciferol-supplemented groups showed significant rise of mean serum 25(OH)D (30.6±7.51 and 28.6±8.41 ng/ml). The expression of LL-37, IFN-γ, IL-4, IL-4δ2 and transcription factors were comparable in the four groups at baseline. Despite significant increase in mean serum 25(OH)D in the cholecalciferol-supplemented groups, their mean mRNA transcripts of LL-37, IFN-γ, IL-4, transcription factors and their IFN-γ/IL-4 and T-bet/GATA-3 ratios were similar to that of calcium and placebo groups.Conclusions:Six months of cholecalciferol/calcium supplementation in young females with VDD do not lead to significant alteration in mRNA expression of LL-37, Th1/Th2 cytokines and their transcription factors.

Polymorphisms at +49A/G and CT60 sites in the 3′ UTR of the CTLA-4 gene and APECED-related AIRE gene mutations analysis in sporadic idiopathic hypoparathyroidism

Autoimmune diseases such as Graves’ disease and type 1 diabetes have been linked with +49A/G and CT60 single nucleotide polymorphisms (SNPs) in the 3′ UTR of the cytotoxic T‐lymphocyte antigen‐4 (CTLA‐4) gene. Both these SNPs are functionally relevant and linked with T‐lymphocyte activation. Hypoparathyroidism is seen in 70% of patients with autoimmune polyendocrinopathy candidiasis ectodermal dystrophy syndrome (APECED). Although calcium sensing receptor autoantibodies (CaSRAb) and generalized activation of T lymphocytes are reported among patients with sporadic idiopathic hypoparathyroidism (SIH), CTLA‐4 gene SNPs and APECED‐related autoimmune regulator (AIRE) gene mutations have not been assessed in them. We studied lead CTLA‐4 gene SNPs and APECED‐related AIRE gene mutations in 73 patients with SIH and 114 healthy subjects. The CTLA‐4 gene SNPs +49A/G in exon 1, CT60A/G in 3′ UTR and −318C/T in the promoter region were genotyped by polymerase chain reaction‐restriction fragment‐length polymorphism (PCR‐RFLP) using BstEII, NcoI and MseI endonucleases, respectively. The APECED‐related AIRE gene mutations, which is R257X (Finn‐major) in exon 6, 4‐bp insertion and 13‐bp deletion in exon 8, and Iranian Jews population ‘Y85C’ mutation in exon 2, were studied by PCR‐RFLP (Taq‐I), PCR and nucleotide sequencing, respectively. CaSRAb were studied by immunoblotting. The frequencies of CTLA‐4 A/A49, A/G49 and G/G49 genotypes in the patients (47.9%, 38.4% and 13.7%) and controls (45.6%, 39.5% and 14.9%, respectively) and the frequencies of CT60 A/A, A/G, and G/G genotypes in the patient (42.4%, 37.0% and 20.6%) and the control (38.6%, 40.4% and 21.0%, respectively) groups were not significantly different. The frequencies of various haplotypes including genetic loci +49A/G and CT60 and frequencies of G alleles at these positions were comparable between patient and the control groups and its presence did not correlate with clinical and biochemical indices of the disease. None of the patients had APECED‐related AIRE gene mutations. Lack of significant difference in the pattern of CTLA‐4 A/G49 and/or CT60A/G genotypes and absence of common APECED syndrome‐related AIRE gene mutations among patients and controls suggest that these sites do not play a role in the development of the SIH.

Prevalence of clinical remission in patients with sporadic idiopathic hypoparathyroidism

Background  Remission of disease activity is a characteristic feature of autoimmune endocrine disorders such as Graves’ disease, Addison’s disease and occasionally in patients with premature ovarian failure. Autoimmunity is also implicated in sporadic idiopathic hypoparathyroidism (SIH) with clinical remission of disease reported in three cases.

Predisposition to vitamin D deficiency osteomalacia and rickets in females is linked to their 25(OH)D and calcium intake rather than vitamin D receptor gene polymorphism

Background   Osteomalacia (OSM) and rickets are widely prevalent in developing countries especially in females. The factors associated with such predisposition are not known.

Protein tyrosine phosphatase non-receptor type 22 (PTPN22) gene R620W variant and sporadic idiopathic hypoparathyroidism in Asian Indians

Recently, a gain of function variant C1858T of the lymphoid‐specific protein tyrosine phosphatase non‐receptor (LYP, PTPN22) gene has been reported to be associated with several autoimmune disorders including Graves’ disease, type 1 diabetes, rheumatoid arthritis and vitiligo. The present study was carried out in 80 patients with sporadic idiopathic hypoparathyroidism (SIH) [43 males and 37 females, mean ± SD age and duration of symptoms 32.5 ± 14.1 years and 6.7 ± 7.2 years (range 1 day to 35 years), respectively] and 193 healthy controls (male : female ratio 91:102, mean ± SD age, 43.1 ± 11.6 years) to assess association of 1858T allele with the disease. Polymerase chain reaction–restriction fragment length polymorphism analysis was performed to genotype C1858T variant. The frequency of occurrence of 1858T allele was 4/160 (2.5%) in SIH and 5/386 (1.3%) in the control alleles (odds ratio 1.95, 95% CI 0.51–7.37). Thus, the present study reveals that 1858T allele is rare (1.3%) in Asian Indians. The trend of higher prevalence of 1858T allele in patients with SIH needs to be studied further in other population with higher rate of the allele to support the autoimmune basis of the disease.