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Summary

  1. Top of page
  2. Summary
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References
  8. Supporting Information

Background

It has been suggested that NLRP1 is involved in susceptibility to a wide range of autoimmune diseases including generalized vitiligo (GV). Genetic polymorphisms in the gene encoding NLRP1 (previously known as NALP1) have previously been shown to be associated with GV and there is speculation about their involvement in the regulation of NLRP1 expression.

Objectives

To explore NLRP1 polymorphisms and investigate their association with NLRP1 mRNA expression and disease activity in patients with GV.

Methods

Polymerase chain reaction (PCR)–restriction fragment length polymorphism and TaqMan single nucleotide polymorphism (SNP) genotyping techniques were used to genotype NLRP1 A/G (rs2670660), T/C (rs6502867) and A/T (rs12150220) polymorphisms in 537 patients with GV and 645 controls in Gujarat. NLRP1 mRNA levels were measured in the whole blood of 122 patients with GV and 175 controls using real-time PCR.

Results

The NLRP1 rs2670660 and rs6502867 polymorphisms were found to be in significant association with GV, minor alleles of these SNPs being prevalent in active cases of GV. The rs12150220 polymorphism was found have a marginal association with GV. The frequency of susceptible haplotype ‘GCT’ was significantly higher in patients with GV and increased the risk of vitiligo twofold. A significant increase in NLRP1 mRNA expression was observed in patients with GV and patients with active GV. NLRP1 mRNA expression was increased in patients with GV with the susceptible GG (rs2670660) and CC (rs6502867) genotypes. Patients with the susceptible GG (rs2670660) and CC (rs6502867) genotypes had early age of onset of GV. Moreover, patients in the age at onset group of 1–20 years showed increased expression of NLRP1 mRNA compared with the older age groups. Female patients showed a significant increase in NLRP1 mRNA and early age at onset of GV compared with male patients.

Conclusions

Our results suggest that NLRP1 rs2670660 and rs6502867 polymorphisms may be genetic risk factors for susceptibility to and progression of GV. The upregulation of NLRP1 mRNA in patients with susceptible genotypes advocates the crucial role of NLRP1 in GV.

Generalized vitiligo (GV) is an acquired, noncontagious disorder in which progressive, patchy loss of pigmentation from the skin, overlying hair and oral mucosa results from autoimmune loss of melanocytes from the involved areas.[1] It is a polygenic, multifactorial disorder involving multiple susceptibility genes and unknown environmental triggers.[2-4] Epidemiological studies have shown frequent family clustering of vitiligo cases, with elevated risk of vitiligo in first-degree relatives and high concordance in monozygotic twins.[4, 5] Approximately one in four patients with vitiligo manifest other autoimmune disorders, which also occur at elevated frequency among the close relatives of patients with vitiligo[5, 6] suggesting that increased risk of vitiligo and other autoimmune disorders in these families has a genetic basis.

More than 30 susceptibility loci have been identified for GV on the basis of genetic linkage and genome-wide association studies (GWAS).[7] GWAS revealed a highly significant association of familial cases of GV with polymorphic variants of the gene encoding NLRP1 (a key regulator of the innate immune system, previously known as NALP1).[8, 9] NLRP1 recognizes pathogen-associated molecular patterns and triggers assembly of the NLRP1 inflammasome, which stimulates inflammatory responses and apoptotic pathways resulting in pathogenesis of vitiligo.[9, 10] The NLRP1 gene is a leucine-rich repeat protein 1 [a member of the NLR family (nucleotide oligomerization domain-like receptor)].[9] It contains a caspase recruitment domain that is known to be the key mediator of apoptosis. High levels of NLRP1 expression in immune cells, particularly T cells and Langerhans cells emphasize the role of NLRP1 in regulation of the immune system.[11]

Genetic association of NLRP1 variants with GV has been shown in populations in the U.S.A., U.K., Romania and Jordan.[9, 12, 13] We have selected three NLRP1 variants, rs2670660, rs12150220 and rs6502867, for the present study as these single nucleotide polymorphisms (SNPs) are among those previously reported to contain high-risk alleles and to be significantly associated with GV.[9, 12, 13] The NLRP1 polymorphisms are also associated with autoimmune conditions such as Addison disease and type 1 diabetes.[14, 15] However, NLRP1 genetic variants have not been assessed for their effect on NLRP1 expression, and onset and progression of GV. Hence, the aims of this study were: (i) to estimate and compare NLRP1 mRNA levels in patients with GV and in unaffected controls; (ii) to determine whether NLRP1 promoter (A/G; rs2670660), structural (A/T; rs12150220) and intronic (T/C; rs6502867) polymorphisms are associated with GV and modulate NLRP1 mRNA levels in these groups; and (iii) to correlate NLRP1 polymorphisms and their mRNA levels with onset and activity of the disease.

Materials and methods

  1. Top of page
  2. Summary
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References
  8. Supporting Information

Study subjects

The study group included 537 patients with GV (including acrofacial vitiligo and vitiligo universalis) who were referred to S.S.G. Hospital, Vadodara and Civil Hospital, Ahmedabad, Gujarat, India (Table S1; see Supporting Information). The diagnosis of vitiligo was clinically based on the presence of depigmented patches on the skin and patients had no other associated autoimmune diseases. The patients were divided into two groups: active GV (existing lesions were spreading and/or new lesions had appeared within the previous 6 months) and stable GV (no increase in lesion size or number within ≥ 6 months to 2 years). Three hundred and ninety-two patients were classified with active GV, and 145 patients were included in the stable GV group (Table S1). A total of 645 ethnically sex-matched unaffected individuals were included as controls (Table S1). None of the healthy individuals or their relatives had any evidence of vitiligo. For the NLRP1 mRNA expression study, 122 patients with GV and 175 healthy age- and sex-matched individuals were involved.

The study plan was approved by the Institutional Ethical Committee for Human Research, Faculty of Science, Maharaja Sayajirao University of Baroda, Vadodara, Gujarat, India. The importance of the study was explained to all participants and written consent was obtained from all patients and controls.

Genomic DNA preparation

Venous blood (5 mL) was collected from the patients and unaffected subjects in K3–ethylenediaminetetraacetic acid-coated vacutainers (BD, Franklin Lakes, NJ, U.S.A.). Genomic DNA was extracted from whole blood using a QIAamp DNA Blood Kit (Qiagen Inc., Valencia, CA, U.S.A.) according to the manufacturer's instructions.

Genotyping of NLRP1 A/G promoter (rs2670660) and T/C intronic (rs6502867) polymorphisms

Polymerase chain reaction (PCR)–restriction fragment length polymorphism (RFLP) was used to genotype NLRP1 A/G (rs2670660) and T/C (rs6502867) polymorphisms (Fig. S1a, b; see Supporting Information). The primers used for the genotyping are listed in Table S2 (see Supporting Information). Amplification was performed using a PTC-100 thermal cycler (MJ Research Inc., Watertown, MA, U.S.A.) according to the protocol: 94 °C for 10 min followed by 35 cycles of 94 °C for 30 s, primer-dependent annealing (Table S2) for 30 s and 72 °C for 30 s.

Restriction enzymes (New England Biolabs, Beverly, MA, U.S.A.) ApoI and Hpy8I were used for digesting amplicons of NLRP1 A/G (rs2670660) and T/C (rs6502867) SNPs, respectively (Table S2). The digested products with a 50-bp or 100-bp DNA ladder (Bioron, Ludwigshafen am Rhein, Germany) were resolved on 2·5% agarose gels stained with ethidium bromide (Fig. S1). More than 10% of the samples were randomly selected for confirmation and the results were 100% concordant (analysis of the chosen samples was repeated by two researchers independently) and also confirmed by DNA sequencing.

Genotyping of NLRP1 A/T structural (rs12150220; Leu155His) polymorphism

The genotyping of the A/T (rs12150220) SNP of NLRP1 was carried out with dual-colour hydrolysis probes (FAM and VIC) using the LightCycler®480 Real-Time PCR protocol (Roche Diagnostics GmbH, Mannheim, Germany) with background-corrected endpoint fluorescence analysis using the TaqMan SNP genotyping assay (Assay ID: C_1600653_10; Life Technologies Corp., Carlsbad, CA, U.S.A.).

Determination of NLRP1 and GAPDH mRNA expression

RNA extraction and cDNA synthesis

Total RNA from whole blood was isolated and purified using a Ribopure™ blood kit (Ambion Inc., Austin, TX, U.S.A.) following the manufacturer's protocol. RNA integrity was verified by 1·5% agarose gel electrophoresis; RNA yield and purity was determined spectrophotometrically at 260/280 nm. cDNA synthesis was performed using 1 μg total RNA and the RevertAid First Strand cDNA Synthesis Kit (Fermentase, Vilnius, Lithuania) according to the manufacturer's instructions.

Real-time polymerase chain reaction

The expression of NLRP1 and GAPDH mRNA was measured with real-time PCR using gene-specific primers (Eurofins, Bangalore, India) as shown in Table S2. Expression of the GAPDH gene was used as a reference. Real-time PCR was performed in duplicates in a 20-μL reaction volume using the LightCycler®480 SYBR Green I Master (Roche Diagnostics) following the manufacturer's instructions. The thermal cycling conditions included an initial activation step at 95 °C for 10 min, followed by 45 cycles of denaturation, annealing and amplification (95 °C for 10 s, 65 °C for 15 s, 72 °C for 20 s). At the end of the amplification phase a melt curve analysis was carried out to check the specificity of the products formed (Fig. S2; see Supporting Information). The PCR cycle at which PCR amplification begins its exponential phase and product fluorescence intensity finally rises above background was considered as the crossing point (CP) or cycle threshold (CT). The ΔCT or ΔCP value was determined as the difference between the cycle threshold of the target gene (NLRP1) and the reference gene (GAPDH). The difference between the two ΔCP values (ΔCP controls and ΔCP patients) was considered as ΔΔCP to obtain the value of fold expression (2ΔΔCp).

Statistical analyses

The distribution of the genotypes and allele frequencies of the NLRP1 polymorphisms for patients and control subjects were compared using the χ2 test with 3 × 2 and 2 × 2 contingency tables, respectively, using Prism 4 software 2003 (GraphPad Software Inc., San Diego, CA, U.S.A.). P < 0·017 was considered statistically significant due to Bonferroni's correction for multiple testing. Haplotype analysis was carried out using SHEsis (http://analysis2.bio-x.cn/myAnalysis.php).[16] The linkage disequilibrium (LD) coefficients D' and r2 values were estimated using the Haploview program version 4.1.[17] Age at onset analysis and relative expression of NLRP1 mRNA in patient and control groups were plotted and analysed with a nonparametric unpaired t-test using Prism 4 software 2003 (GraphPad Software Inc.).

Results

  1. Top of page
  2. Summary
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References
  8. Supporting Information

Analysis of association between NLRP1 (A/G; rs2670660) promoter polymorphism and susceptibility to generalized vitiligo

PCR-RFLP for the NLRP1 A/G promoter polymorphism yielded a 227-bp undigested product corresponding to the ‘G’ allele, and 179-bp and 48-bp digested products corresponding to the ‘A’ allele (Fig. S1a). The NLRP1 A/G promoter polymorphism was found to be in significant association with patients with GV (P < 0·0001) when genotypes were compared using the χ2 test 3 × 2 contingency table (Table 1). In particular, the minor allele ‘G’ was prevalent in the GV group compared with the control group and this was consistent with a susceptibility effect in the GV group [51·0% vs. 42·0%, P < 0·0001, odds ratio (OR) 1·46, 95% confidence interval (CI) 1·24–1·72] (Table 1). Both control and patient groups were found to be in Hardy–Weinberg equilibrium (HWE) for this polymorphism (= 0·062 and = 0·135, respectively) (Table 1).

Table 1. Association studies for NLRP1 gene A/G (rs2670660) promoter, T/C (rs6502867) intron and A/T (rs12150220; Leu155His) structural polymorphisms in patients with generalized vitiligo (GV) and controls from Gujarat
SNPPatients with GV, n (freq.) (= 537)Controls, n (freq.) (= 645)P for associationP for HWEOdds ratio (95% CI)
  1. SNP, single nucleotide polymorphism; HWE, Hardy–Weinberg equilibrium; CI, confidence interval; (P), patients; (C), controls. aPatients with GV vs. controls using χ2 test with 3 × 2 contingency table; bpatients with GV vs. controls using χ2 test with 2 × 2 contingency table; values are significant at P ≤ 0·017 due to Bonferroni's correction.

rs2670660 (A/G)
Genotype     
AA136 (0·25)229 (0·36)< 0·0001a

0·135 (P)

0·062 (C)

 
AG251 (0·47)291 (0·45)
GG150 (0·28)125 (0·19)
Allele    
A523 (0·49)749 (0·58)< 0·0001b1·46 (1·24–1·72)
G551 (0·51)541 (0·42)
rs6502867 (T/C)
Genotype     
TT199 (0·37)312 (0·48)< 0·0001a0·071 (P) 0·096 (C) 
TC239 (0·44)260 (0·40)
CC99 (0·18)73 (0·12)
Allele    
T637 (0·59)884 (0·69)< 0·0001b1·49 (1·26–1·77)
C437 (0·41)406 (0·31)
rs12150220 (A/T)
Genotype     
AA298 (0·55)400 (0·62)0·074a0·728 (P) 0·052 (C) 
AT202 (0·38)205 (0·32)
TT37 (0·07)40 (0·06)
Allele    
A788 (0·73)1005 (0·78)0·012b1·28 (1·06–1·55)
T286 (0·27)285 (0·22)

Analysis based on the stage of progression of GV revealed that the frequency of the minor ‘G’ allele was increased in the active GV group (56·0% vs. 42·0%, P < 0·0001) compared with the control group (Table 2). However, no significant difference was observed for the distribution of ‘G’ allele between stable GV and control groups (= 0·235) (Table 2). Interestingly, the ‘G’ allele was prevalent in patients with active GV compared with stable GV (56·0% vs. 38·0%, P < 0·0001). In addition, the sex-based analysis suggested significant association of the ‘G’ allele with female patients compared with male patients (55·0% vs. 47·0%, = 0·007, OR 1·40, 95% CI 1·09–1·78) (Table 3) and the ‘G’ allele was consistent with a susceptibility effect in female patients.

Table 2. Association studies for NLRP1 gene A/G (rs2670660) promoter, T/C (rs6502867) intron and A/T (rs12150220; Leu155His) structural polymorphisms in patients with active and stable generalized vitiligo (GV) from Gujarat
SNPActive vitiligo, n (freq.) (= 392)Stable vitiligo, n (freq.) (= 145)Controls, n (freq.) (= 645)P for associationP for HWEOdds ratio (95% CI)
  1. SNP, single nucleotide polymorphism; HWE, Hardy–Weinberg equilibrium; CI, confidence interval; (AV), active vitiligo; (SV), stable vitiligo; (C), controls. aActive vitiligo vs. stable vitiligo; bactive vitiligo vs. controls; cstable vitiligo vs. controls; values are significant at P ≤ 0·017 due to Bonferroni's correction.

rs2670660 (A/G)
Genotype      
AA77 (0·20)59 (0·40)229 (0·36)

< 0·0001a

< 0·0001b

0·467c

0·686 (AV)

0·268 (SV)

0·062 (C)

 
AG189 (0·48)62 (0·43)291 (0·45)
GG126 (0·32)24 (0·17)125 (0·19)
Allele    

0·48a (0·36–0·63)

0·56b (0·47–0·67)

1·18c (0·91–1·54)

A343 (0·44)180 (0·62)749 (0·58)

< 0·0001a

< 0·0001b

0·23c

G441 (0·56)110 (0·38)541 (0·42)
rs6502867 (T/C)
Genotype      
TT129 (0·33)70 (0·48)312 (0·48)

0·004a

< 0·0001b

0·676c

0·366 (AV)

0·094 (SV)

0·096 (C)

 
TC184 (0·47)55 (0·38)260 (0·40)
CC79 (0·20)20 (0·14)73 (0·12)
Allele    

0·63a (0·47–0·84)

0·59b (0·49–0·71)

0·94c (0·72–1·24)

T442 (0·56)195 (0·67)884 (0·69)

0·001a

< 0·0001b

0·676c

C342 (0·44)95 (0·33)406 (0·31)
rs12150220 (A/T)
Genotype      
AA216 (0·55)82 (0·57)400 (0·62)

0·843a

0·081b

0·464c

0·995 (AV)

0·496 (SV)

0·052 (C)

 
AT150 (0·38)52 (0·36)205 (0·32)
TT26 (0·07)11 (0·07)40 (0·06)
Allele    

0·90a (0·66–1·22)

0·74b (0·60–0·91)

0·83c (0·62–1·11)

A582 (0·74)216 (0·74)1005 (0·78)

0·532a

0·006b

0·215c

T202 (0·26)74 (0·26)285 (0·22)
Table 3. Association studies for NLRP1 gene A/G (rs2670660) promoter, T/C (rs6502867) intron and A/T (rs12150220; Leu155His) structural polymorphisms in male and female patients with generalized vitiligo
SNPMale patients, n (freq.) (= 237)Female patients, n (freq.) (= 300)P for associationP for HWEOdds ratio (95% CI)
  1. SNP, single nucleotide polymorphism; HWE, Hardy–Weinberg equilibrium; CI, confidence interval; (M) male patients; (F), female patients. aMale patients vs. female patients using χ2 test with 3 × 2 contingency table; bmale patients vs. female patients using χ2test with 2 × 2 contingency table; values are significant at P ≤ 0·017 due to Bonferroni's correction.

rs2670660 (A/G)
Genotype     
AA74 (0·31)62 (0·20)0·017a

0·091 (M)

0·771 (F)

 
AG105 (0·44)146 (0·50)
GG58 (0·25)92 (0·30)
Allele    
A253 (0·53)270 (0·45)0·007b1·40 (1·09–1·78)
G221 (0·47)330 (0·55)
rs6502867 (T/C)
Genotype     
TT105 (0·44)94 (0·31)0·003a

0·220 (M)

0·311 (F)

 
TC99 (0·42)140 (0·47)
CC33 (0·14)66 (0·22)
Allele    
T309 (0·65)328 (0·55)0·001b1·55 (1·21–1·99)
C165 (0·35)272 (0·45)
rs12150220 (A/T; Leu155His)
Genotype     
AA136 (0·57)162 (0·54)0·729a

0·586 (M)

0·984 (F)

 
AT85 (0·36)117 (0·39)
TT16 (0·07)21 (0·07)
Allele    
A357 (0·75)441 (0·74)0·527b1·10 (0·83–1·45)
T117 (0·25)159 (0·26)

Analysis of association between NLRP1 (T/C; rs6502867) intron polymorphism and susceptibility to generalized vitiligo

PCR-RFLP for the NLRP1 T/C intron polymorphism yielded a 216-bp undigested product corresponding to the ‘C’ allele, and 125-bp and 91-bp digested products corresponding to the ‘T’ allele (Fig. S1b). The NLRP1 T/C polymorphism was found to be in significant association with patients with GV (P < 0·0001) when genotypes were compared using the χ2 test 3 × 2 contingency table (Table 1). In particular, the minor allele ‘C’ was prevalent in the GV group compared with the control group and was consistent with a susceptibility effect with GV (41·0% vs. 31·0%, P < 0·0001, OR 1·49, 95% CI 1·26–1·77) (Table 1). Both control and patient groups were found to be in HWE for this polymorphism (= 0·096 and = 0·071, respectively) (Table 1).

Analysis based on the stage of progression of GV revealed that the frequency of the minor ‘C’ allele was increased in the active GV group (44·0% vs. 31·0%, P < 0·0001) compared with the control group (Table 2). However, no significant difference was observed in the distribution of the ‘C’ allele between the stable GV and control groups (= 0·676) (Table 2). Interestingly, the ‘C’ allele was prevalent in patients with active GV compared with stable GV (44·0% vs. 33·0%, = 0·001). In addition, sex-based analysis indicated a significant association of the ‘C’ allele with female patients and the ‘C’ allele was consistent with a susceptibility effect (45·0% vs. 35·0%, = 0·001, OR 1·55, 95% CI 1·21–1·99) (Table 3).

Analysis of association between NLRP1 (A/T; rs12150220) structural polymorphism and susceptibility to generalized vitiligo

Real-time PCR-based TaqMan SNP genotyping method was carried out using dual-colour hydrolysis probes labelled with FAM (for ‘T’ allele) and VIC (for ‘A’ allele) fluorophores for the NLRP1 A/T (rs12150220) polymorphism, which yielded the three genotypes (AA homozygous, AT heterozygous and TT homozygous) as identified by scatter plots using background-corrected endpoint fluorescence analysis (Fig. S3; see Supporting Information).

The NLRP1 A/T polymorphism was found to be in marginal association with patients with GV as suggested by different allele frequencies for the polymorphism and the polymorphism was consistent with a susceptibility effect with the GV group (27·0% vs. 22·0%, = 0·012, OR 1·28, 95% CI 1·06–1·55); however, genotype frequencies did not differ for the polymorphism between patients and controls (= 0·074) (Table 1). Both control and patient groups were found to be in HWE for this polymorphism (= 0·052 and = 0·728, respectively) (Table 1).

Analysis based on the stage of progression of GV revealed that the minor ‘T’ allele frequency was increased in the active GV group (26·0% vs. 22·0%, = 0·006) compared with the control group (Table 2). There was no significant difference observed in the distribution of the ‘T’ allele between the active and stable GV groups (= 0·532), and the stable GV and control groups (= 0·215) (Table 2). Also, no significant difference was observed in ‘T’ allele frequency between male and female patients (= 0·527) (Table 3).

Linkage disequilibrium and haplotype analyses

LD analysis revealed that the three polymorphisms investigated in the NLRP1 gene were in low LD association [A/G : T/C (D = 0·08, r2 = 0·003); A/G : A/T (D = 0·21, r2 = 0·02); T/C : A/T (D = 0·09, r2 = 0·01)]. A haplotype evaluation of the three polymorphic sites was performed and the estimated frequencies of the haplotypes differed significantly between patients with GV and controls (global P-value = 1·54 × 10−8) (Table 4).

Table 4. Distribution of haplotype frequencies for NLRP1 gene A/G (rs2670660) promoter, T/C (rs6502867) intron and A/T (rs12150220; Leu155His) structural polymorphisms among patients with generalized vitiligo (GV) and controls
Haplotype [A/G (rs2670660), T/C (rs6502867) and A/T (rs12150220)]Patients with GV, n (freq.) (= 966)Controls, n (freq.) (= 1066)P for association P (global) Odds ratio (95% CI)
  1. CI, confidence interval. Frequencies (freq.) < 0·03 in controls and cases dropped and ignored in the analysis.

A C A142·63 (0·15)170·64 (0·16)0·4441·54 × 10−80·91 (0·72–1·16)
A C T41·91 (0·04)56·25 (0·05)0·3270·82 (0·54–1·23)
A T A196·76 (0·20)312·78 (0·29)3·42 × 10−60·62 (0·50–0·76)
A T T73·70 (0·08)85·33 (0·08)0·7590·95 (0·69–1·32)
G C A100·25 (0·10)69·26 (0·07)0·0021·67 (1·21–2·30)
G C T92·22 (0·09)51·85 (0·05)3·94 × 10−52·07 (1·45–2·94)
G T A205·37 (0·21)235·32 (0·22)0·6650·95 (0·77–1·18)
G T T112·17 (0·12)84·57 (0·08)0·0101·53 (1·13–2·05)

Interestingly, the frequency of susceptible haplotype ‘GCT’ containing the minor alleles of all the three polymorphisms was significantly higher in patients with GV compared with controls (9·5% vs. 4·9%, = 3·94 × 10−5) and increased the risk of vitiligo by twofold (OR 2·07, 95% CI 1·45–2·94) (Table 4). Also two other haplotypes, ‘GCA’ and ‘GTT’, were significantly increased in patients with GV compared with controls (= 0·002 and = 0·01, respectively). The ancestral ‘ATA’ haplotype was more frequently observed in the control group compared with the patient group (29·3% vs. 20%, = 3·42 × 10−6) (Table 4).

Effect of NLRP1 polymorphisms on age at onset of generalized vitiligo

When age at onset of the disease was correlated with the NLRP1 A/G (rs2670660) promoter genotypes, patients with susceptible GG genotypes showed early onset of the disease compared with the AA and AG genotypes (P < 0·0001 and = 0·011, respectively) (Fig. 1a). Moreover, patients with the AG genotype showed early onset of the disease compared with the AA genotypes (= 0·003) (Fig. 1a).

image

Figure 1. Age at onset of the disease in patients with generalized vitiligo (GV). (a) Comparison of age at onset of the disease with respect to the NLRP1 A/G (rs2670660) polymorphism in 537 patients with GV. Patients with the GG genotype showed early age at onset compared with AA and AG genotypes. Patients with the AG genotype showed early age at onset compared with the AA genotype. (b) Comparison of age at onset of the disease with respect to the NLRP1 T/C (rs6502867) polymorphism in 537 patients with GV. Patients with the CC genotype showed early age at onset compared with the TT and TC genotypes. Patients with the TC genotype showed early age at onset compared with the TT genotype. (c) Comparison of age at onset of the disease with respect to the NLRP1 A/T (rs12150220) polymorphism in 537 patients with GV. No significant difference was observed in age at onset between patients with the AA and TT genotypes and the AT genotype. Also, patients with the AT genotype did not show significant difference in age at onset of disease compared with the TT genotype. (d) Comparison of age at onset of the disease with respect to sex differences in 237 male patients and 300 female patients with GV. Female patients showed early age at onset compared with male patients.

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Analysis of age at onset with NLRP1 T/C (rs6502867) genotypes revealed that patients with susceptible CC genotypes showed early onset of the disease compared with the TT and TC genotypes (P < 0·0001 and = 0·0003, respectively) (Fig. 1b). Moreover, patients with genotype TC showed early onset of the disease compared with TT genotypes (= 0·032) (Fig. 1b).

However, patients with NLRP1 (rs12150220) AA genotypes did not show any significant difference for the age at onset of the disease compared with the TT (= 0·163) and AT genotypes (= 0·635) (Fig. 1c). Also, patients with the AT genotype did not show significant difference in age at onset of disease compared with the TT genotype (= 0·357) (Fig. 1c). Interestingly, when male and female patients with GV were analysed for age at onset of the disease, female patients showed significant early onset compared with male patients (P < 0·0001) (Fig. 1d).

Relative gene expression of NLRP1 in patients with generalized vitiligo and controls

Comparison of the findings showed a significant increase in expression of NLRP1 mRNA in 122 patients with GV compared with 175 unaffected controls after normalization with GAPDH expression as indicated by mean ΔCp values (P < 0·0001) (Fig. 2a). In particular, patients with GV showed 7·6-fold higher expression of NLRP1 mRNA compared with controls (Fig. 2b).

image

Figure 2. Relative expression of the gene NLRP1 in controls and patients with generalized vitiligo (GV). (a) Expression of NLRP1 mRNA in 175 controls and 122 patients with GV. Patients showed increased NLRP1 mRNA expression compared with controls. (b) Fold expression of NLRP1 mRNA in 122 patients with GV and 175 controls showed 7·6-fold higher expression in patients compared with controls, as determined by the 2∆∆Cp method. (c) Expression of NLRP1 mRNA with respect to NLRP1 A/G (rs2670660) promoter polymorphism in 175 controls and 122 patients with GV. Patients with the GG and AG genotypes showed increased NLRP1 mRNA expression compared with controls. Patients with the AA genotype showed no significant difference in NLRP1 mRNA expression compared with controls. (d) Expression of NLRP1 mRNA with respect to the NLRP1 T/C (rs6502867) intron polymorphism in 175 controls and 122 patients with GV. Patients with the CC and TC genotypes showed increased NLRP1 mRNA expression compared with controls. Patients with the TT genotype showed no significant difference in NLRP1 mRNA expression compared with controls. (e) Expression of NLRP1 mRNA with respect to the NLRP1 A/T (rs12150220) intron polymorphism in 175 controls and 122 patients with GV. Patients with the TT, AT and AA genotypes showed no significant difference in NLRP1 mRNA expression compared with controls. (f) Expression of NLRP1 mRNA with respect to progression of the disease in 91 patients with active vitiligo and 31 patients with stable vitiligo. Patients with active vitiligo showed increased NLRP1 mRNA expression compared with controls. (g) Expression of NLRP1 mRNA with respect to sex differences in 52 male patients and 70 female patients with GV. Female patients showed increased NLRP1 mRNA expression compared with controls. (h) Expression of NLRP1 mRNA with respect to different age at onset groups in 122 patients with GV. Patients in the age at onset group 1–20 years showed increased NLRP1 mRNA expression compared with age at onset groups 21–40, 41–60 and 61–80 years. (a, c–h) Expression of NLRP1 mRNA indicated by mean ∆Cp.

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Correlation of NLRP1 mRNA with the investigated polymorphisms

Further, the expression of NLRP1 mRNA was analysed with respect to A/G (rs2670660) promoter genotypes (Fig. 2c). Interestingly, NLRP1 mRNA expression was significantly higher in patients with a susceptible GG genotype compared with controls (= 0·001). Also, patients with the AG genotype showed increased NLRP1 mRNA expression compared with controls (= 0·007); however, no significant difference was observed in NLRP1 mRNA expression in patients compared with controls with AA genotypes (= 0·102).

The genotype–phenotype correlation for T/C (rs6502867) showed significant increase in expression of NLRP1 mRNA in patients with CC and TC genotypes compared with controls (= 0·020, = 0·006, respectively); however, no significant difference was observed in NLRP1 mRNA expression in patients compared with controls with the TT genotype (= 0·121) (Fig. 2d). The genotype–phenotype analysis for the A/T (rs12150220) SNP did not show any significant difference in NLRP1 mRNA expression between patients and controls (= 0·082, = 0·162, = 0·160) (Fig. 2e).

Effect of NLRP1 mRNA expression on disease progression

We also checked the effect of NLRP1 mRNA expression on progression of the disease, i.e. active and stable cases (Fig. 2f). Interestingly, patients with active GV showed a significant increase in expression of NLRP1 mRNA compared with the patients with stable GV (= 0·021). Patients with active GV showed a 5·3-fold higher expression of NLRP1 mRNA compared with the patients with stable GV (Fig. S4a; see Supporting Information). Moreover, the susceptibility for the disease was also analysed based on sex differences and we found that female patients showed significantly higher NLRP1 mRNA expression compared with male patients (= 0·001) (Fig. 2g). In particular, female patients showed a 6·0-fold higher expression of NLRP1 mRNA compared with controls (Fig. S4b).

When NLRP1 mRNA expression was monitored in different age at onset groups of patients, patients in the age at onset group 1–20 years showed significantly higher expression of NLRP1 mRNA compared with the age at onset groups 21–40, 41–60 and 61–80 years (= 0·044, = 0·038 and = 0·029, respectively) (Fig. 2h).

Discussion

  1. Top of page
  2. Summary
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References
  8. Supporting Information

NLRP1 is known to be involved in inflammation and apoptosis.[18, 19] It modulates the response of cells towards proinflammatory cytokines such as interleukin (IL)-1β, interferon (IFN)-γ and tumour necrosis factor (TNF)-α. Moreover, it has been suggested that melanocyte death is mediated by apoptosis in the context of autoimmunity, and cytokines such as IFN-γ, TNF-α and IL-1β can initiate apoptosis.[20] Recently, we showed that increased TNF-α and its promoter polymorphisms correlate with disease progression and higher susceptibility towards vitiligo.[21] Overexpression of the NLRP1 gene induces apoptosis in cells and even transient overexpression of NLRP1 in in vitro cultured cells was sufficient to induce apoptosis.[22] NLRP1 is a component in the active assembly of inflammasomes.[23] The NLRP1 inflammasome activates caspase-1, which in turn activates proinflammatory cytokines IL-1β and IL-18.[24]

Recently, a functional analysis of NLRP1 haplotypes associated with vitiligo and autoimmunity found an increase in IL-1β processing; however, NLRP1 mRNA and protein levels were not altered by the predominant high-risk haplotype, suggesting that altered function of the corresponding multivariant NLRP1 polypeptide predisposes to autoimmune diseases by activation of the NLRP1 inflammasome.[25] This haplotype however involves coding sequence variants, not the promoter and intronic SNPs reported in this study. Here, we report the association of promoter and intronic SNPs with NLRP1 expression; hence the two studies are compatible. Our recent study also suggested that IL1B mRNA expression is increased in patients with GV compared with controls (N.C. Laddha, M. Dwivedi, M.S. Mansuri, M. Singh, D. Krishnan, H.H. Patel, N. Agarwal, A.M. Shah and R. Begum, unpublished data). IL-1β can act on lymphocytes and upregulates IL-2 receptor expression, prolonging survival of T cells, enhancing antibody production by B cells and increasing B-cell proliferation.[26, 27] IL-1β and IL-18 also play a crucial role in driving the differentiation and amplification of T-helper (Th)17 and Th1 cells, respectively.[28-30] Thus, IL-1β and IL-18 amplify T- and B-cell responses and might serve as a crucial link in translating NLRP1 activation into adaptive immune responses.

NLRP1 is expressed at low levels in all cells but is expressed at high levels in blood mononuclear cells and immune cells, particularly granulocytes, monocytes, T lymphocytes and Langerhans cells. Immunohistochemical studies have shown high NLRP1 expression in the epidermis within Langerhans cells[22] suggesting the involvement of NLRP1 in skin autoimmunity. NLRP1 could also trigger or enhance autoimmunity as crosstalk occurs between melanocytes and NLRP1-positive Langerhans cells.[31] Previously, several SNPs in NLRP1 have been studied in connection with susceptibility to GV; however, only a few of them achieved a significant association.[9, 12, 13] The functional significance of these SNPs is not yet known, but they are proposed to have a regulatory effect on NLRP1 expression. Studies of white patients with GV from the U.K., the U.S.A. and Romania[9, 12] confirmed the genetic association of GV with NLRP1 variants, which contain at least two independent risk signals, one tagged by SNP rs2670660 and another tagged by SNPs rs2670660 and rs8182352. A significant association with GV of two SNPs in the NLRP1 extended promoter region (rs1008588 and rs2670660) was also shown in an Arab population.[13] The present study focuses on identification of genetic variants of NLRP1 that were shown to be significantly associated with susceptibility to GV in earlier studies. Interestingly, we found a significant association of NLRP1 rs2670660 (A/G) and rs6502867 (T/C) SNPs with susceptibility to GV in the Gujarat population; however, in the current study GV was associated with the minor allele (‘C’) at SNP rs6502867, whereas GV is associated with the major allele (‘T’) in white subjects;[12] also there was marginal association with another SNP, rs12150220 (A/T), which is associated in white subjects.[9] Moreover, the difference in association of the NLRP1 rs6502867 allelic variant in the Gujarat population from that of white subjects suggests a recombination event that is well correlated with the ethnic difference (i.e. occurred early in the divergence of the races) and also suggests that this SNP is not in itself causal for GV but tightly linked to a causal variant. Also, the difference from the highly significant association for rs12150220 SNP in white subjects may be explained by this SNP having recombined with the causal variant(s) in a proportion of the Gujarat population.

Furthermore, the ‘G’ allele of promoter polymorphism (A/G; rs2670660), which may be involved in regulation of NLRP1 expression, was found to be prevalent in GV, especially in active cases, which signifies that NLRP1 also modulates disease progression. Moreover, the minor allele ‘G’ was prevalent in female patients compared with male patients suggesting a genetic predisposition for GV in female subjects. Another SNP rs6502867 (T/C), present in the intronic region of NLRP1, was also found to be in significant association with GV, active cases of GV and with female patients with GV suggesting its crucial role in vitiligo pathogenesis. Furthermore, the higher frequency of the susceptible haplotype ‘GCT’ in patients with GV increased the risk of vitiligo twofold indicating the combined effect of the three susceptible alleles of NLRP1 SNPs in GV.

Recently, Deo et al.[32] showed a significant increase in NLRP1 mRNA expression in patients with GV; however, the sample size was very small. The present study also found a significant increase in expression of NLRP1 mRNA (7·6-fold) in patients with GV compared with controls. The expression of NLRP1 mRNA was also increased (5·3-fold) in active cases compared with stable cases of GV suggesting a crucial role for NLRP1 in the progression of GV. Also, female patients had increased expression of NLRP1 mRNA (6·0-fold) and exhibited early age at onset of GV compared with male patients with GV supporting the fact that female subjects are more susceptible to autoimmune diseases such as GV.[33-35] The NLRP1 mRNA expression was also increased in patients with an early onset of GV compared with those of late onset suggesting the important role of NLRP1 in the early phase of the disease. In support of this, age at onset analysis with different genotypes of NLRP1 polymorphisms, A/G (rs2670660) and T/C (rs6502867), revealed that patients harbouring susceptible genotypes had an early onset of GV compared with those with the ancestral genotypes.

The genotype–phenotype correlation for the NLRP1 A/G (rs2670660) SNP revealed increased expression of NLRP1 mRNA in patients with GG and AG genotypes compared with controls indicating that the ‘G’ allele may be involved in the increased NLRP1 expression. Moreover, genotype–phenotype analysis of the NLRP1 T/C (rs6502867) SNP showed increased expression of NLRP1 mRNA in patients with CC and TC genotypes compared with controls, suggesting the important role of the ‘C’ allele in increased NLRP1 expression. However, the nonsynonymous A/T (rs12150220) SNP did not reveal any significant correlation of its genotypes with NLRP1 expression.

The exact functional consequences of these polymorphic variants remain to be determined. The complexity of NLRP1 regulation and its various putative effector mechanisms (inflammation, apoptosis, etc.) raise several interesting possibilities for how the sequence variations in NLRP1 might impact immune system function. The present study for the first time reports that NLRP1 rs2670660 and rs6502867 SNPs are associated with increased NLRP1 mRNA expression; however, further investigations are needed to elaborate the mechanistic insights that might reveal functional consequences of NLRP1 variants in GV and other autoimmune disorders. The study also revealed differences in association of NLRP1 allelic variants compared with white patients, as GV was associated with the opposite allele at SNP rs6502867 in the Gujarat population and there was marginal association with another SNP (rs12150220) associated with GV in white subjects. Moreover, the study also emphasizes the prevalence of high-risk alleles of NLRP1 SNPs (rs2670660 and rs6502867) and increased expression of NLRP1 mRNA in female patients and those with active vitiligo. In addition, age at onset analysis revealed an association of high-risk alleles of NLRP1 SNPs (rs2670660 and rs6502867) and increased expression of NLRP1 mRNA with early age at onset in patients with GV.

Recently, we showed a positive association of HLA-A*33:01, HLA-B*44:03 and HLA-DRB1*07:01 in patients with vitiligo from North India and Gujarat suggesting an autoimmune link of vitiligo in these cohorts.[36] We have also shown that the three most significant class II region SNPs – rs3096691 (just upstream of NOTCH4), rs3129859 (just upstream of HLA-DRA), and rs482044 (between HLA-DRB1 and HLA-DQA1) – are associated with GV in an Indian population.[37] The genotype–phenotype correlation of TNF, CTLA4, IL4, IFNG and MYG1 (C12orf10) gene polymorphisms also supports the autoimmune pathogenesis of vitiligo in the Gujarat population, whereas our earlier studies on CAT, GPX, MBL2, ACE and PTPN22 polymorphisms did not show significant association.[21, 38-45] In addition, our recent study indicates that an imbalance of the CD4+/CD8+ ratio and natural regulatory T cells in frequency and function might be involved in the T cell-mediated pathogenesis of GV and its progression.[46]

In conclusion, our findings suggest that the increased NLRP1 expression in patients with GV could result, at least in part, from variations at the genetic level. The study also emphasizes the influence of NLRP1 on disease progression, onset and sex bias for developing GV. More detailed studies regarding the role of NLRP1 in the occurrence of vitiligo may prove to be useful for the development of effective preventive/ameliorative therapies.

Acknowledgments

  1. Top of page
  2. Summary
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References
  8. Supporting Information

R.B. thanks ICMR, New Delhi, India, DBT, New Delhi, India, and GSBTM, Gandhinagar, Gujarat, India. N.C.L. thanks the Council of Scientific and Industrial Research, New Delhi for awarding SRF.

References

  1. Top of page
  2. Summary
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References
  8. Supporting Information

Supporting Information

  1. Top of page
  2. Summary
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References
  8. Supporting Information
FilenameFormatSizeDescription
bjd12467-sup-0001-FigS1.tifimage/tif2064KFig S1. Polymerase chain reaction–restriction fragment length polymorphism (PCR-RFLP) analysis of NLRP1 (previously NALP1) A/G (rs2670660) and C/T (rs6502867) polymorphisms. (a) PCR-RFLP analysis of NLRP1 A/G (rs2670660) promoter polymorphism on 2·5% agarose gel electrophoresis: lanes 4 and 12, homozygous (AA) genotypes; lanes 6–9, heterozygous (AG) genotypes; lanes 1–3, 5, 10 and 11, homozygous (GG) genotypes; lane M, 100-bp DNA ladder. (b) PCR-RFLP analysis of NLRP1 C/T (rs6502867) intron polymorphism on 2·5% agarose gel electrophoresis: lanes 1, 2, 4–6 and 8, homozygous (CC) genotypes; lanes 3 and 7, heterozygous (CT) genotypes; lane M, 50-bp DNA ladder.
bjd12467-sup-0002-FigS2.tifimage/tif3453KFig S2. Melt curve analysis of NLRP1 (previously NALP1) and GAPDH showing specific amplification.
bjd12467-sup-0003-FigS3.tifimage/tif3179KFig S3. TaqMan endpoint fluorescence analysis for NLRP1 (previously NALP1) A/T (rs12150220) using dual-colour hydrolysis probes (FAM and VIC) by LightCycler®480 Real-Time PCR protocol. The three genotypes identified as AA, AT and TT based on fluorescence with channel 465–510 (FAM for ‘T’ allele) and channel 536–580 (VIC for ‘A’ allele). A no-template control (NTC) was used with the SNP genotyping assay.
bjd12467-sup-0004-FigS4.tifimage/tif3083KFig S4. Fold expression of NLRP1 (previously NALP1) mRNA in patients with generalized vitiligo (GV). (a) Fold expression of NLRP1 mRNA in 31 patients with stable and 91 patients with active vitiligo. Patients with active vitiligo showed 5·3-fold higher expression compared with patients with stable vitiligo, as determined by the 2∆∆Cp method. (b) Fold expression of NLRP1 mRNA in 52 male and 70 female patients with vitiligo. Female patients showed a 6·0-fold higher expression compared with male patients, as determined by the 2∆∆Cp method.
bjd12467-sup-0005-TableS1-S2.docWord document37K

Table S1. Demographic characteristics of generalized vitiligo (GV) patients and unaffected controls.

Table S2. Primers and restriction enzymes used for NLRP1 gene A/G promoter (rs2670660) and T/C (rs6502867) intron SNPs genotyping and gene expression analyses.

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