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Published ahead of print on January 10, 2008, doi:10.1164/rccm.200705-781OC
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American Journal of Respiratory and Critical Care Medicine Vol 177. pp. 712-719, (2008)
© 2008 American Thoracic Society
doi: 10.1164/rccm.200705-781OC

Original Article

A Genetic Variation in Inositol Polyphosphate 4 Phosphatase A Enhances Susceptibility to Asthma

Mamta Sharma1, Jyotsna Batra1, Ulaganathan Mabalirajan1, Shilpy Sharma1, Rana Nagarkatti1, Jyotirmoi Aich1, Surendra K. Sharma2, Pramod V. Niphadkar3 and Balaram Ghosh1

1 Molecular Immunogenetics Laboratory, Institute of Genomics and Integrative Biology, Delhi, India; 2 Division of Pulmonary and Critical Care Medicine, Department of Medicine, All India Institute of Medical Sciences, Delhi, India; and 3 Asthma and Allergy Centre, Mumbai, India

Correspondence and requests for reprints should be addressed to Dr. Balaram Ghosh, Ph.D., Molecular Immunogenetics Laboratory, Institute of Genomics and Integrative Biology, Mall Road, Delhi 110007, India. E-mail: bghosh{at}igib.res.in


   ABSTRACT
TOP
 ABSTRACT
AT A GLANCE COMMENTARY
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Rationale: Microarray data from mouse studies have identified a number of genes to be differentially expressed in allergen-sensitized mice lungs.

Objectives: Taking leads from these datasets, we attempted to identify novel genes associated with atopic asthma in humans.

Methods: We performed family-based genetic association analysis on selected markers within or in proximity of 21 human homologs of genes short-listed from ovalbumin-sensitized mouse studies in the Gene Expression Omnibus database of the National Center for Biotechnology Information. Family-based and case-control studies were undertaken for fine mapping and functional variation analysis of INPP4A (inositol polyphosphate 4 phosphatase type I). Western blot analysis was performed to analyze INPP4A protein stability from human platelets.

Measurements and Main Results: Our genetic association studies of 21 human genes in 171 trios led to the identification of a biallelic repeat (rs3217304) in INPP4A, associated with atopic asthma (P = 0.009). Further studies using additional three single nucleotide polymorphisms (SNPs), +92031A/T, +92344C/T, and +131237C/T, and two microsatellite markers, D2S2311 and D2S2187, revealed significant genetic associations with loci +92031A/T (P = 0.0012) and +92344C/T (P = 0.004). A nonsynonymous SNP, +110832A/G (Thr/Ala), present within a sequence enriched with proline, glutamic acid, serine, and threonine (PEST), in proximity of these two loci, showed a significant association with atopic asthma (P = 0.0006). The association results were also replicated in an independent cohort of 288 patients and 293 control subjects (P = 0.004). PEST score and Western blot analyses indicated a functional role of this SNP in regulating INPP4A protein stability.

Conclusions: In our study, INPP4A was identified as a novel asthma candidate gene, whereby the +110832A/G (Thr/Ala) variant affected its stability and was significantly associated with asthma.

Key Words: asthma • gene • INPP4A • single nucleotide polymorphism



   AT A GLANCE COMMENTARY
TOP
 ABSTRACT
 AT A GLANCE COMMENTARY
METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Scientific Knowledge on the Subject
Signaling cascades involving PI3K–Akt pathway genes SHIP and PTEN have been found to be associated with asthma. However, no study involving the terminal enzyme in the pathway, INPP4A, has been undertaken in asthma and allergic disorders.

What This Study Adds to the Field
Using a genomewide candidate gene approach, we demonstrate the association of INPP4A with atopic asthma.

 
Atopic asthma is a chronic inflammatory lung disorder characterized by recurrent breathing problems in response to various allergens and environmental stimuli and is associated with substantial morbidity, mortality, and person-days loss (1, 2). Both inflammation and airway remodeling are found to be important components of complex asthma pathology, in which mast cells, T cells, eosinophils, epithelial cells, and platelets play key roles (3). Interactions among these cells induce many signaling events, including regulation of the phosphatidyl–inositol–Akt pathway (4).

Although familial aggregation of asthma is well documented (5, 6), no unique model delineates its mode of inheritance. Many asthma-associated genes have been identified using candidate gene, linkage, and positional cloning approaches (7–11); nevertheless, more remain to be identified (12, 13). Although a candidate gene approach does not lead to the identification of novel disease-associated genes, genomewide scans are comprehensive but time consuming and expensive (14). Here, we attempted to identify new asthma-associated genes by a genomewide candidate approach using a combination of bioinformatic and experimental methods. The Gene Expression Omnibus (GEO) at the National Center for Biotechnology Information (NCBI) is the largest repository for high-throughput molecular abundance data, primarily gene expression data generated through microarray experiments (15, 16). Comparative analysis of the datasets on microarray gene expression studies in allergen-induced mouse models led us to identify 21 potential asthma candidate genes. By undertaking genetic studies on these genes in humans, we identified INPP4A (inositol polyphosphate 4 phosphatase type I) as a potential asthma-associated gene (Figure 1). INPP4A is a magnesium-independent phosphatase, which is expressed in human brain, platelets, megakaryocytes, and Jurkat T cells (17). Although there are no reports on the expression of INPP4A in human mast cells or basophils, our preliminary results indicated its expression in an eosinophilic cell line (EOL-1) and A549 and THP-1 (monocytic leukemia) cell lines (B.G. and J.A., unpublished data). Also, the expression of Inpp4b, but not of Inpp4a, was detected in mouse mast cells (18). In any event, this enzyme is suggested to negatively regulate the phosphatidylinositol 3 kinase (PI3K) pathway by modulating intracellular phosphatidyl inositol 3,4 bisphosphate (PtdIns[3,4]P2) levels. PI3K–Akt signaling is critical for various asthma-associated pathophysiologic pathways, including platelet activation (4, 19, 20). Activated platelets release several inflammatory mediators and growth factors that aggravate the asthma condition (21). Earlier experiments in mice identified two other negative regulators of the PI3K–Akt pathway, Src homology 2–containing inositol phosphatase (SHIP) (22) and phosphatase and tensin homolog deleted on chromosome 10 (PTEN) (23), which are associated with asthma phenotype. However, no studies so far have been undertaken showing involvement of INPP4A in asthma and allergic disorders.


Figure 1
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  		Figure 1. Outline sketch of the working strategy to identify novel asthma-associated candidate genes using genomewide candidate gene approach. GDS = gene dataset; PEST = proline, glutamic acid, serine, and threonine; SNP = single nucleotide polymorphism.

 
This is the first study to establish a genetic association of INPP4A with atopic asthma. Moreover, by fine mapping of INPP4A, we identified association of a nonsynonymous polymorphism, A+110832G (Thr/Ala), with atopic asthma, which was found to alter the stability of the encoded {alpha}3 protein spliceoform (17) in platelets. The implication of these results in the context of asthma pathogenesis involving INPP4A and PI3K is discussed. Some of the results of this study have been previously reported in the form of an abstract (24).


   METHODS
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 ABSTRACT
 AT A GLANCE COMMENTARY
 METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
GEO Dataset Analysis and Marker Selection
We searched the GEO database for experiments involving allergen-induced asthma and identified three gene datasets: GDS347, GDS348, and GDS349 (15). The genes showing more than threefold differences in at least five samples in any one dataset, and found to be present in asthma/atopy-linked syntenic regions in humans, were short-listed (n = 69) (Figure 1; Table E1 in the online supplement). Complete genes and flanking region sequences were downloaded from the NCBI nucleotide database and were run through RepeatMasker (www.repeatmasker.org/cgi-bin/WEBRepeatMasker; Institute of Systems Biology, Seattle, WA) to identify simple repeats.

Sample Collection
Cases with atopic asthma and their parents (n = 171 trios for initial screening of 21 genes, n = 277 trios for the fine mapping of INPP4A) and an independent case-control (patients, nP = 288; control subjects, nc = 293) cohort of Indo-Aryan origin were recruited from various hospitals in North India (Table 1). Ethical approval was obtained from the Institute of Genomics and Integrative Biology review board. Written, informed consent was obtained from all participating individuals. Subjects with family history of asthma were diagnosed for asthma by a physician according to the guidelines of the National Asthma Education and Prevention Program (Expert Panel Report 2) (25) and were examined for a self-reported history of breathlessness and wheezing (26). Probands met the following criteria: FEV1/FVC of less than 70% at the time of attack and improvement by a bronchodilator, positive skin prick test to at least 1 of the 15 common environmental allergens, and undergoing asthma therapy. Healthy volunteers (referred to as normal control subjects) were recruited from the general population (case-matched geographical area) and were skin prick test negative, had normal pulmonary function test values, and answered negatively to the screening questionnaire for respiratory symptoms and met the criteria of having no symptoms or history of allergic disease symptoms and not currently smoking (within past 3 yr). Blood samples were obtained and DNA was isolated by a salting out method as described previously (26). Total serum IgE levels were estimated using ELISA (Bethyl Labs, Montgomery, TX).


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  		TABLE 1. CLINICAL AND PHYSIOLOGIC CHARACTERISTICS OF THE PROBANDS IN THE TRIO STUDY AND PATIENTS AND CONTROLS IN THE CASE-CONTROL STUDY

 
Genotyping of Repetitive Sequences in or around the 21 Short-listed Genes and INPP4A
Polymerase chain reaction was carried out using a FAM/HEX (fluorescein amidite, hexachloro-fluorescein)-labeled forward primer and an unlabeled reverse primer (Table E2). These samples were subsequently genotyped by electrophoresis on ABI Prism 3100 Genetic Analyzer (Applied Biosystems [ABI], Foster City, CA) as per the manufacturer's instructions. Fragment lengths were determined using the GeneScan/GeneMapper version 3.7 (ABI) as described (26).

INPP4A SNP Identification and Genotyping
DNA from 16 individuals (normal control subjects) was sequenced to identify SNPs in INPP4A (Figure E1). Coding polymorphisms were identified by cDNA sequencing using primer sets CDS1–CDS4 (Table E2). SNPs at positions A+92031T (rs3769712), C+92344T (rs3769710), C+131237T (rs10201079), and A+110832G (rs2278206) were genotyped using SNaPshot ddNTP Primer Extension Kit (ABI).

PESTfind Analysis of the AF368319 Transcript Protein {alpha}3
PESTfind, a web-based algorithm (http://www.at.embnet.org/embnet/tools/bio/PESTfind/; European Molecular Biology Network, Vienna, Austria), was run for the {alpha}3 variant of INPP4A protein with threonine or alanine residue at amino acid position 604.

Platelet Isolation, Stimulation, and Western Blot Analysis
Platelets were isolated from peripheral blood of nine normal healthy individuals (three individuals for each genotype: AA, GG, and AG for A+110832G polymorphism) and stimulated with 2 µM ionomycin in platelet suspension buffer for 5 and 10 minutes, respectively (27). Western blot was performed using goat polyclonal antibody sc-12315 (Santa Cruz Biotechnology, Santa Cruz, CA) and anti-goat horseradish peroxidase antibody. Densitometry scanning of the bands was done using AlphaImager (Alpha Innotech Corp., San Leandro, CA).

Plasma RANTES Level Measurement
To determine RANTES (regulated upon activation, normal T-cell expressed and secreted) levels in the plasma, we used the RANTES ELISA kit (R&D Systems, Minneapolis, MN) as per the manufacturer's instructions.

Statistical Analysis
An extended transmission disequilibrium test (ETDT) (28) (ETDT25; http://www.mds.qmw.ac.uk/statgen/dcurtis/software.html, London Statistical Genetics Group, London, UK), TDT/s-TDT program 1.1 (29, 30), and a family-based association test (FBAT) (31) (http://www.biostat.harvard.edu/~fbat/default.html) were used to evaluate the genetic association of various loci with asthma. A haplotype-based association test (HBAT) was carried out using HBAT option of FBAT. Each SNP locus was evaluated for Hardy-Weinberg equilibrium and pairwise linkage disequilibrium between each pair of SNP loci was calculated using De Finneti (http://ihg.gsf.de/cgi-bin/hw/hwa1.pl) and Haploview (32), respectively. Armitage trend test was used to analyze the association in case-control studies. Multiple logistic regression analysis was performed to see the effect of age and sex, if any. Differences in haplotype frequencies in cases and control subjects were compared using a Monte Carlo approach as implemented in CLUMP2.3 (33) (London Statistical Genetics Group). A P value less than 0.007 (Bonferroni-adjusted {alpha} = 0.05/7 = 0.007) was considered significant.


   RESULTS
TOP
 ABSTRACT
 AT A GLANCE COMMENTARY
 METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Leads from GEO Databases and Preliminary Evidence of Association of INPP4A with Asthma
Using bioinformatics-based comparative analysis of the three GEO mouse datasets (GDS347, GDS348, and GDS349), we identified 69 differentially expressed genes present in regions that were linked with asthma/atopy (Table E1). We hypothesized that these genes could be responsible for allergen sensitivity or resistance, and might contribute toward atopic asthma. To identify the associated gene by high-throughput screening, repeat polymorphisms were used as markers because of their high heterozygosity and informativeness in family-based association studies. The genes in which polymorphic repeats were identified were therefore also selected for high-throughput association studies. Because the aim was to identify new candidate genes, the genes that have previously been shown to be associated with asthma were not included in this study. Thus, we included 21 genes (human homologs) in our study using a family-based association test on 171 case–parent trios for simple repeats as markers (Table E3) and found a simple repeat, rs3217304 in intron 11 (+99097), of INPP4A to be associated with atopic asthma (P < 0.05).

Fine Mapping of INPP4A and Identification of a Nonsynonymous A+110832G (Thr/Ala) Polymorphism Associated with Asthma
Because INPP4A seemed to be a potential novel asthma candidate, we attempted to genetically tag the INPP4A gene to confirm its association with asthma. Five microsatellite repeat markers, besides the biallelic simple repeat rs3217304 (S3) used for initial genetic screening, were also identified within and around the gene, but only two (D2S2311 [M1] and D2S2187 [M2]) were found to be polymorphic and selected for genetic studies (Figure 2A). We also identified 15 SNPs in six segments, which were selected to encompass the INPP4A 74.9–142.7-kb genetic region, by sequencing the genomic DNA of 16 individuals. Of these, nine SNPs (A+74647G, T+74854C, A+92031T, C+92344T, C+92817T, C+131237T, G+131595A, T+133683C, and A+133699C) had a minor allele frequency greater than 5% (Figure E1). On the basis of their linkage disequilibrium pattern and amenability to genotyping, three SNPs (A+92031T [S1], C+92344T [S2], and C+131237T [S5]) were selected for further studies in the initial 171 and the additional 106 trios (total n = 277), and also in an independent case-control (np = 288 and nc = 293 for cases and controls, respectively) study (Table 1) (see Figure E2a). We observed a significant association of A+92031T (S1), C+92344T (S2), and the +99097 biallelic repeat (S3) with asthma in the initial 171 trios (see Table E4) as well as in the total set of 277 trios. The allele A of A+92031T (105 transmissions/63 nontransmissions, P = 0.0012), allele C of C+92344T (58 transmissions/31 nontransmissions, P = 0.004), and allele 2 of the +99097 biallelic repeat (102 transmissions/67 nontransmissions, P = 0.007) were found to be associated with atopic asthma. The association for the S1 was significant even after Bonferroni correction (P = 0.008). The microsatellite repeat D2S2311 also showed marginal significance (Table 2). Similar results were obtained in our case-control studies except for S2 (Table 3, Figure E3). This suggested the possibility of a functional polymorphism in vicinity or in linkage disequilibrium with A+92031T (S1) and +99097 biallelic repeat (S3) loci.


Figure 2
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  		Figure 2. (A) Schematic diagram showing the markers and single nucleotide polymorphisms studied. M1, M2, S3, S1, S2, S4, and S5 are D2S2311, D2S2187, rs3217304, A+92031T, C+92344T, A+110832G, and C+131237T, respectively. (B) Isoforms of INPP4A {alpha} resulting from alternative splicing in the genetic region encompassing the exons 17, 18, and 19. These variants encode three protein spliceoforms ({alpha}1, {alpha}2, and {alpha}3) in different tissues. INPP4A-{alpha}1 is a 106-kD major form expressed in human and mouse brain, INPP4A-{alpha}2 is a 102-kD minor form expressed in mouse brain, and INPP4A {alpha}3 is a 110-kD major form expressed in human platelets, MEG-01 megakaryocytic cells, and Jurkat T-cells. (C) INPP4A {alpha} variants showing the PEST (proline, glutamic acid, serine, and threonine) sequence and their PEST scores as analyzed using PESTfind software. The {alpha}1 variant has a PEST site in the region encoded by exons 18 and 19 with a PEST score of +5.36; the PEST site is absent in the {alpha}2 variant, whereas the {alpha}3 variant has a PEST site in the additional 40 amino acid region encoded by extended exon 17 with a PEST score of +7.49. Substitution of threonine by alanine (A+110832G; underlined) at amino acid position 604 in the {alpha}3 variant results in a reduced PEST score of +4.95.

 

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  		TABLE 2. TEST OF ASSOCIATION FOR INDIVIDUAL LOCI GENE VARIANTS OF INPP4A WITH ATOPIC ASTHMA IN CASE–PARENT TRIOS*

 

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  		TABLE 3. GENOTYPE DISTRIBUTION OF INPP4A POLYMORPHISMS IN ATOPIC SUBJECTS WITH ASTHMA AND NORMAL SUBJECTS IN AN INDIAN POPULATION

 
To identify any coding SNP/SNPs in INPP4A, we sequenced the cDNA prepared from the leukocytes of 16 individuals. This led to the identification of a nonsynonymous Thr/Ala SNP A+110832G (S4) in the transcript AF368319 present within the PEST region of the encoded {alpha}3 spliceoform of the INPP4A protein (Figure 2B). PEST sequences are proline-glutamate/aspartate-serine/threonine–rich motifs, which are recognized by calpain proteases and thus play a pivotal role in protein degradation. This SNP is located at distances 18.8 and 11.7 kb from loci S1 and S3, respectively. The family-based association study revealed a significant overtransmission of allele A (118 transmissions/71 nontransmissions, P = 0.0006) to the affected children (Table 2). Similarly, frequency of the GG genotype was found to be more in control subjects when compared with the patients (P = 0.004; odds ratio, 0.13; 95% confidence interval, 0.11–0.75) in case-control studies.

Haplotypic Association of INPP4A Variants with Atopic Asthma
To further the understanding of INPP4A gene variants in asthma, we constructed haplotypes encompassing intronic SNP S1, microsatellite repeat S3, and the nonsynonymous SNP S4 (associated with asthma in both the study designs). The analysis of haplotypes showed an overall significant association using HBAT (P = 0.002, {chi}2 = 11.88, df = 2). Haplotype analysis, collapsing columns with small expected values together, in the case-control studies also showed a significant association (P < 0.001, {chi}2 = 22.6, df = 2). Individually, the haplotype A_2_A was found to be positively associated with atopic asthma (P < 0.001 in case-control studies and trios), whereas haplotype T_1_G was negatively associated (P = 0.001 in case-control and P = 0.005 in trios) (Table 4).


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  		TABLE 4. TEST OF ASSOCIATION OF INPP4A HAPLOTYPES WITH ATOPIC ASTHMA IN 277 TRIOS AND IN CASE-CONTROL STUDIES*

 
A+110832G (Thr/Ala) Polymorphism Increases INPP4A Protein Stability in Platelets
Because the A+110832G (Thr/Ala) polymorphism was present in the region encoding the PEST sequence of the INPP4A {alpha}3 variant, to elucidate the functional implication of this polymorphism PESTfind analysis was performed. The PESTfind algorithm allows rapid and objective identification of PEST motifs in protein target sequences and produces a score ranging from –50 to +50. PESTfind uses a default threshold of +5.0 for discriminating weak from potential PEST motifs. PESTfind analysis showed a significant decrease in the PEST score (+7.49 to +4.95) and therefore increased protein stability after Thr to Ala substitution (Figure 2C).

Because the INPP4A {alpha}3 protein variant is known to be expressed as a major form in platelets, its stability in platelets from normal healthy individuals of different genotypes at A+110832G (S4) was checked. The platelets were stimulated with the Ca2+ ionophore, ionomycin, and the degradation of INPP4A was checked by Western blot. Ionomycin increases Ca2+ influx and results in activation of calcium-activated calpain proteases and therefore degradation of proteins containing PEST motifs. Protein from platelets of individuals with the AA genotype was found to be more susceptible to degradation as seen by nearly 53% degradation within 5 minutes of stimulation (P = 0.0009), whereas the protein from individuals with the GG genotype was less susceptible (~8% degradation, P = 0.61) (Figure 3). Also, the basal levels of INPP4A protein from individuals with AA genotypes were found to be lower, suggesting poor stability in vivo as well.


Figure 3
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  		Figure 3. (A) Western blot analysis for INPP4A and β-tubulin in platelets isolated from human blood (a representative picture of three independent experiments) having AA (lanes 1, 2, and 3), GG (lanes 4, 5, and 6), and AG (lanes 7, 8, and 9) genotypes. Lanes 1, 4, and 7 represent platelets without stimulation; lanes 2, 5, and 8 represent platelets stimulated with 2 µM ionomycin for 5 minutes; lanes 3, 6, and 9 represent platelets stimulated with 2 µM ionomycin for 10 minutes. (B) Relative intensity of INPP4A protein with respect to mean of β-actin and β-tubulin in unstimulated and stimulated platelets as estimated by spot densitometry scanning of Western blot data. (C) The log10 plasma RANTES levels with respect to different genotypes of A+110832G single nucleotide polymorphism. "N" represents number of individuals in each group. RANTES = regulated upon activation, normal T-cell expressed and secreted.

 
A+110832G (Thr/Ala) Polymorphism Is Associated with Plasma RANTES Levels
RANTES, a CC-chemokine with a potent chemoattractant activity, is secreted by lymphocytes and constitutively present in platelet {alpha}-granules and released upon platelet activation (21, 34). To demonstrate the functional significance of INPP4A genetic variants, we measured the RANTES levels in the plasma of 115 individuals and correlated them with the A+110832G (Thr/Ala) SNP of INPP4A (Figure 3C). We found a significant association of plasma RANTES level with A+110832G (P = 0.0007 after correction for age and sex), where AA individuals were found to have high mean plasma RANTES levels, when compared with AG or GG individuals.


   DISCUSSION
TOP
 ABSTRACT
 AT A GLANCE COMMENTARY
 METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
We identified a novel asthma-associated gene, INPP4A, by integrating the extracted information from microarray datasets together with human genome sequence and experimental genomic and functional approaches. The enzyme INPP4A dephosphorylates and inactivates PtdIns(3,4)P2, an important messenger in the PI3K–Akt pathway, which is associated with proliferation of airway smooth muscle (35), platelet activation (4), mast cell degranulation (20), and bronchial hyperresponsiveness in a mouse model of asthma (23) (Figure 4). In fact, being the terminal enzyme for the PtdIns(3,4,5)P3 degradation pathway, INPP4A could be more critical in determining the signaling through the Akt pathway in comparison to SHIP and PTEN, which have already been characterized as prime asthma-associated genes (22, 23).


Figure 4
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  		Figure 4. Role of INPP4A in asthma pathogenesis: PtdIns(3,4)P2, an important messenger in the PI3K–Akt pathway, is associated with proliferation of airways smooth muscle, platelet activation, mast cell degranulation, and bronchial hyperresponsiveness. INPP4A enzyme dephosphorylates PtdIns(3,4)P2 and thus inactivates the PI3K–Akt signaling pathway. INPP4A was found to be inactivated by calpain-mediated proteolysis in stimulated and activated platelets, which accounts for the accumulation of PtdIns(3,4)P2 in these cells (27). Thus, the levels of INPP4A could affect the asthma status (this study). BHR = bronchial hyperresponsiveness; PtdIns(3,4)P2 = phosphatidyl inositol 3,4 bisphosphate; PtdIns(4,5)P2 = phosphatidyl inositol 4,5 bisphosphate; PtdInst(3,4,5)P3 = phosphatidyl inositol 3,4,5 triphosphate; PTEN = phosphatase and tensin homolog deleted on chromosome 10; SHIP = Src homology 2–containing inositol phosphatase.

 
Because INPP4A seemed to be a potential novel asthma candidate, we attempted to identify its genetic association with asthma. Asthma is a complex genetic disorder; due to small effects of individual genes, a family-based study may fail to detect significant association even when it exists (36, 37). Thus, we recruited additional families to impart greater power to our analysis. Furthermore, we replicated our results in an independently recruited case-control cohort. We have tried to rule out the possibility of population stratification in our case-control studies by the following: (1) recruiting ethnicity-matched patients and controls by taking the comprehensive information about their geographical area, ethnic background, and migration status (Indo-Aryan origin), and (2) performing multiple logistic regression analysis to see the effects of age and/or sex on observed association. Thus, it is very unlikely that our results are due to stratification or an inherent statistical bias. Our fine mapping study on the INPP4A genetic variants led to the identification of a nonsynonymous SNP, A+110832G (Thr/Ala), which was found to be associated with asthma using both the study designs. The nonsynonymous SNP, A+110832G (Thr/Ala), is present in the additional 40 amino acid domain of the {alpha}3 spliceoform of INPP4A (Figure 2B), a major form expressed in platelets (17). In vitro studies using PI3K inhibitors, wortmannin, and LY294002 suggest an important role for the PI3 kinases in regulating a broad range of functional platelet responses, including primary platelet adhesion, cytoskeletal remodeling, and platelet aggregation (4). Platelets accumulate in the pulmonary microvasculature and release inflammatory mediators upon activation in provoked or spontaneous asthma attacks (21). We observed that the A+110832G (Thr/Ala) polymorphism is present in the region encoding the PEST domain of INPP4A {alpha}3. Activation of platelets is known to be associated with degradation of INPP4A by calpain-mediated proteolysis and accumulation of PtdIns(3, 4)P2 substrate (27). PESTfind analysis of the {alpha}3 protein 604Thr/Ala variants revealed a significant reduction in the PEST score, making the region a poor PEST site. Thus, the threonine to alanine substitution might reduce the susceptibility of INPP4A {alpha}3 to calpain proteases, and in turn may result in less signaling by the PI3K pathway, alleviating the asthma condition (Figure 4). This hypothesis was validated by analyzing proteins isolated from the platelets of the individuals with varying genotypes at A+110832G. The protein from individuals with the AA genotype was found to be less stable than from GG and AG genotypes. Also, the basal levels of INPP4A protein from individuals with AA genotypes were found to be lower (data not shown), suggesting poor stability in vivo as well. Association of higher plasma RANTES levels with AA genotype further strengthens our hypothesis (Figure 3C). The absence of any other functional polymorphisms in the exonic region of INPP4A leads us to believe that the A+110832G (Thr/Ala) polymorphism is primarily responsible for a loss of function phenotype in asthmatic individuals. However, very little is known about the regulation of transcription or translation, formation of spliceoforms, or stability in the asthmatic patient's lungs. Future studies are needed to determine the relationship of INPP4A and the pathogenesis of atopic asthma.

In summary, bioinformatics-based analysis of microarray datasets suggested a protective role of INPP4A due to its reduced mRNA expression in ovalbumin-challenged mice. In addition, genetic fine mapping studies combined with protein expression analyses identified a nonsynonymous SNP, A+110832G, that alters the stability of INPP4A protein in platelets. Thus, this is the first study demonstrating the involvement of INPP4A in mediating atopic asthma and may open new avenues for therapeutic intervention by modulating the PI3K–Akt pathway.


   Acknowledgments
 
The authors thank their collaborating physicians for helping them in sample collection. They also acknowledge the expert help provided by Prof. Partha P. Majumder, Indian Statistical Institute, Kolkata, on statistical analysis. Help extended by Mr. Amrendra Kumar, Mr. Rajshekhar Chatterjee, Ms. Deepti Mann, and Ms. Atoshi Banerjee is greatly acknowledged. The authors also thank all patients, their family members, and healthy volunteers for participating in this study.


   FOOTNOTES
 
Supported by the Task Force Project SMM0006 of Council of Scientific and Industrial Research (CSIR), Government of India. M.S., J.B., and S.S. acknowledge CSIR for their fellowships.

Present address for R.N. is Virginia Bioinformatics Institute, Virginia Tech, Blacksburg, VA 24060.

This article has an online supplement, which is accessible from this issue's table of contents at www.atsjournals.org

Originally Published in Press as DOI: 10.1164/rccm.200705-781OC on January 10, 2008

Conflict of Interest Statement: None of the authors has a financial relationship with a commercial entity that has an interest in the subject of this manuscript.

Received in original form May 28, 2007; accepted in final form January 8, 2008


   REFERENCES
TOP
 ABSTRACT
 AT A GLANCE COMMENTARY
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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