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1.  Haplotype Analysis of the Promoter Region of Phosphodiesterase Type 8B (PDE8B) in Correlation with Inactivating PDE8B Mutation and the Serum Thyroid-Stimulating Hormone Levels 
Thyroid  2010;20(4):363-367.
Background
Human phosphodiesterase (PDE) type 8B (PDE8B) is located at 5q14.1 and is known as the PDE with the highest affinity to cAMP. We recently described a family with bilateral micronodular adrenocortical disease that was apparently caused by an inactivating PDE8B mutation (H305P). As a result of a genome-wide study, a strong association between six polymorphic variants in the PDE8B promoter and serum levels of the thyroid-stimulating hormone (TSH) has been recently reported. Despite an extended analysis of the regions surrounding 5q14.1, no other potential genetic variants that could be responsible for the associated TSH levels were found.
Methods
In this study, we genotyped by polymerase chain reaction the described six polymorphic variants in the PDE8B promoter in the family with micronodular adrenocortical disease and inactivating PDE8B mutation and analyzed their correlation with individual TSH values in the family members.
Results
We observed complete segregation between the reported association and individual TSH values in the family we studied. Haplotype analysis showed that the haplotype associated with the high TSH levels is different from the one that segregated with H305P, suggesting that the mutation most probably has arisen on an allele independent of the high TSH-associated allele.
Conclusions
The proposed mechanism by which PDE8B may influence TSH levels is through control of cAMP signaling. Our analysis revealed separate segregation of an inactivating PDE8B allele from the high-TSH-allele and showed low TSH levels in persons who carry an inactivating PDE8B allele. These data suggest that, indeed, PDE8B may be involved in regulation of TSH levels.
doi:10.1089/thy.2009.0260
PMCID: PMC2867554  PMID: 20373981
2.  Whole-exome sequencing of a pedigree segregating asthma 
BMC Medical Genetics  2012;13:95.
Background
Despite the success of genome-wide association studies for asthma, few, if any, definitively causal variants have been identified and there is still a substantial portion of the heritability of the disease yet to be discovered. Some of this “missing heritability” may be accounted for by family-specific coding variants found to be segregating with asthma.
Methods
To identify family-specific variants segregating with asthma, we recruited one family from a previous study of asthma as reporting multiple asthmatic and non-asthmatic children. We performed whole-exome sequencing on all four children and both parents and identified coding variants segregating with asthma that were not found in other variant databases.
Results
Ten novel variants were identified that were found in the two affected offspring and affected mother, but absent in the unaffected father and two unaffected offspring. Of these ten, variants in three genes (PDE4DIP, CBLB, and KALRN) were deemed of particular interest based on their functional prediction scores and previously reported function or asthma association. We did not identify any common risk variants segregating with asthma, however, we did observe an increase in the number of novel, nonsynonymous variants in asthma candidate genes in the asthmatic children compared to the non-asthmatic children.
Conclusions
This is the first report applying exome sequencing to identify asthma susceptibility variants. Despite having sequenced only one family segregating asthma, we have identified several potentially functional variants in interesting asthma candidate genes. This will provide the basis for future work in which more families will be sequenced to identify variants across families that cluster within genes.
doi:10.1186/1471-2350-13-95
PMCID: PMC3563469  PMID: 23046476
Asthma; Whole-exome sequencing; PDE4DIP; CBLB; KALRN
3.  Association between ORMDL3, IL1RL1 and a deletion on chromosome 17q21 with asthma risk in Australia 
Genome-wide association studies followed by replication provide a powerful approach to map genetic risk factors for asthma. We sought to search for new variants associated with asthma and attempt to replicate the association with four loci reported previously (ORMDL3, PDE4D, DENND1B and IL1RL1). Genome-wide association analyses of individual single nucleotide polymorphisms (SNPs), rare copy number variants (CNVs) and overall CNV burden were carried out in 986 asthma cases and 1846 asthma-free controls from Australia. The most-associated locus in the SNP analysis was ORMDL3 (rs6503525, P=4.8 × 10−7). Five other loci were associated with P<10−5, most notably the chemokine CXC motif ligand 14 (CXCL14) gene (rs31263, P=7.8 × 10−6). We found no evidence for association with the specific risk variants reported recently for PDE4D, DENND1B and ILR1L1. However, a variant in IL1RL1 that is in low linkage disequilibrium with that reported previously was associated with asthma risk after accounting for all variants tested (rs10197862, gene wide P=0.01). This association replicated convincingly in an independent cohort (P=2.4 × 10−4). A 300-kb deletion on chromosome 17q21 was associated with asthma risk, but this did not reach experiment-wide significance. Asthma cases and controls had comparable CNV rates, length and number of genes affected by deletions or duplications. In conclusion, we confirm the association between asthma risk and variants in ORMDL3 and identify a novel risk variant in IL1RL1. Follow-up of the 17q21 deletion in larger cohorts is warranted.
doi:10.1038/ejhg.2010.191
PMCID: PMC3060316  PMID: 21150878
whole-genome; gene; atopy; heterogeneity; structural; IKZF3
4.  Phosphodiesterase 4B is essential for TH2-cell function and development of airway hyperresponsiveness in allergic asthma 
Background
Cyclic AMP (cAMP) signaling modulates functions of inflammatory cells involved in the pathogenesis of asthma, and type 4 cAMP-specific phosphodiesterases (PDE4s) are essential components of this pathway. Induction of the PDE4 isoform PDE4B is necessary for Toll-like receptor signaling in monocytes and macrophages and is associated with T cell receptor/CD3 in T cells; however, its exact physiological function in the development of allergic asthma remains undefined.
Objectives
We investigated the role of PDE4B in the development of allergen-induced airway hyperresponsiveness (AHR) and TH2-driven inflammatory responses.
Methods
Wild-type and PDE4B−/− mice were sensitized and challenged with ovalbumin and AHR measured in response to inhaled methacholine. Airway inflammation was characterized by analyzing leukocyte infiltration and cytokine accumulation in the airways. Ovalbumin-stimulated cell proliferation and TH2 cytokine production were determined in cultured bronchial lymph node cells.
Results
Mice deficient in PDE4B do not develop AHR. This protective effect was associated with a significant decrease in eosinophils recruitment to the lungs and decreased TH2 cytokine levels in the bronchoalveolar lavage fluid. Defects in T-cell replication, TH2 cytokine production, and dendritic cell migration were evident in cells from the airway-draining lymph nodes. Conversely, accumulation of the TH1 cytokine IFN-γ was not affected in PDE4B−/− mice. Ablation of the orthologous PDE4 gene PDE4A has no impact on airway inflammation.
Conclusion
By relieving a cAMP-negative constraint, PDE4B plays an essential role in TH2-cell activation and dendritic cell recruitment during airway inflammation. These findings provide proof of concept that PDE4 inhibitors with PDE4B selectivity may have efficacy in asthma treatment.
doi:10.1016/j.jaci.2010.08.014
PMCID: PMC3002752  PMID: 21047676
Asthma; PDE4B; TH2 cytokines; airway hyperresponsiveness; airway inflammation; cAMP signaling
5.  Modelling of oedemous limbs and venous ulcers using partial differential equations 
Background
Oedema, commonly known as tissue swelling, occurs mainly on the leg and the arm. The condition may be associated with a range of causes such as venous diseases, trauma, infection, joint disease and orthopaedic surgery. Oedema is caused by both lymphatic and chronic venous insufficiency, which leads to pooling of blood and fluid in the extremities. This results in swelling, mild redness and scaling of the skin, all of which can culminate in ulceration.
Methods
We present a method to model a wide variety of geometries of limbs affected by oedema and venous ulcers. The shape modelling is based on the PDE method where a set of boundary curves are extracted from 3D scan data and are utilised as boundary conditions to solve a PDE, which provides the geometry of an affected limb. For this work we utilise a mixture of fourth order and sixth order PDEs, the solutions of which enable us to obtain a good representative shape of the limb and associated ulcers in question.
Results
A series of examples are discussed demonstrating the capability of the method to produce good representative shapes of limbs by utilising a series of curves extracted from the scan data. In particular we show how the method could be used to model the shape of an arm and a leg with an associated ulcer.
Conclusion
We show how PDE based shape modelling techniques can be utilised to generate a variety of limb shapes and associated ulcers by means of a series of curves extracted from scan data. We also discuss how the method could be used to manipulate a generic shape of a limb and an associated wound so that the model could be fine-tuned for a particular patient.
doi:10.1186/1742-4682-2-28
PMCID: PMC1198260  PMID: 16078992
6.  Functional Rescue of Degenerating Photoreceptors in Mice Homozygous for a Hypomorphic cGMP Phosphodiesterase 6 b Allele (Pde6bH620Q) 
Purpose
Approximately 8% of autosomal recessive retinitis pigmentosa (RP) cases worldwide are due to defects in rod-specific phosphodiesterase PDE6, a tetramer consisting of catalytic (PDE6α and PDE6β) and two regulatory (PDE6γ) subunits. In mice homozygous for a nonsense Pde6brd1 allele, absence of PDE6 activity is associated with retinal disease similar to humans. Although studied for 80 years, the rapid degeneration Pde6brd1 phenotype has limited analyses and therapeutic modeling. Moreover, this model does not represent human RP involving PDE6B missense mutations. In the current study the mouse missense allele, Pde6bH620Q was characterized further.
Methods
Photoreceptor degeneration in Pde6bH620Q homozygotes was documented by histochemistry, whereas PDE6β expression and activity were monitored by immunoblotting and cGMP assays. To measure changes in rod physiology, electroretinograms and intracellular Ca2+ recording were performed. To test the effectiveness of gene therapy, Opsin::Pde6b lentivirus was subretinally injected into Pde6bH620Q homozygotes.
Results
Within 3 weeks of birth, the Pde6bH620Q homozygotes displayed relatively normal photoreceptors, but by 7 weeks degeneration was largely complete. Before degeneration, PDE6β expression and PDE6 activity were reduced. Although light-/dark-adapted total cGMP levels appeared normal, Pde6bH620Q homozygotes exhibited depressed rod function and elevated outer segment Ca2+. Transduction with Opsin::Pde6b lentivirus resulted in histologic and functional rescue of photoreceptors.
Conclusions
Pde6bH620Q homozygous mice exhibit a hypomorphic phenotype with partial PDE6 activity that may result in an increased Ca2+ to promote photoreceptor death. As degeneration in Pde6bH620Q mutants is slower than in Pde6brd1 mice and can be suppressed by Pde6b transduction, this Pde6bH620Q model may provide an alternate means to explore new treatments of RP.
doi:10.1167/iovs.07-1422
PMCID: PMC2715364  PMID: 18658088
7.  Phenotypic and genetic heterogeneity in a genome-wide linkage study of asthma families 
Background
Asthma is a complex genetic disease with more than 20 genome-wide scans conducted so far. Regions on almost every chromosome have been linked to asthma and several genes have been associated. However, most of these associations are weak and are still awaiting replication.
Methods
In this study, we conducted a second-stage genome-wide scan with 408 microsatellite markers on 201 asthma-affected sib pair families and defined clinical subgroups to identify phenotype-genotype relations.
Results
The lowest P value for asthma in the total sample was 0.003 on chromosome 11, while several of the clinical subsets reached lower significance levels than in the overall sample. Suggestive evidence for linkage (p = 0.0007) was found for total IgE on chromosomes 1, 7 and again on chromosome 11, as well as for HDM asthma on chromosome 12. Weaker linkage signals could be found on chromosomes 4 and 5 for early onset and HDM, and, newly described, on chromosome 2 for severe asthma and on chromosome 9 for hay fever.
Conclusions
This phenotypic dissection underlines the importance of detailed clinical characterisations and the extreme genetic heterogeneity of asthma.
doi:10.1186/1471-2466-5-1
PMCID: PMC548502  PMID: 15634351
8.  Significant linkage to chromosome 12q24.32–q24.33 and identification of SFRS8 as a possible asthma susceptibility gene 
Thorax  2006;61(10):874-879.
Background
Asthma is a complex genetic disorder. Many studies have suggested that chromosome 12q harbours a susceptibility gene for asthma and atopy. Linkage on chromosome 12q24.21–q24.33 was investigated in 167 Danish families with asthma.
Methods
A two step procedure was used: (1) a genome‐wide scan in one set of families followed by (2) fine scale mapping in an independent set of families in candidate regions with a maximum likelihood score (MLS) of ⩾1.5 in the genome‐wide scan. Polymorphisms in a candidate gene in the region on 12q24.33 were tested for association with asthma in a family based transmission disequilibrium test.
Results
An MLS of 3.27 was obtained at 12q24.33. The significance of this result was tested by simulation, resulting in a significant empirical genome‐wide p value of 0.018. To our Knowledge, this is the first significant evidence for linkage on chromosome 12q, and suggests a candidate region distal to most previously reported regions. Three single nucleotide polymorphisms in splicing factor, arginine/serine‐rich 8 (SFRS8) had an association with asthma (p⩽0.0020–0.050) in a sample of 136 asthmatic sib pairs. SFRS8 regulates the splicing of CD45, a protein which, through alternative splice variants, has an essential role in activating T cells. T cells are involved in the pathogenesis of atopic diseases such as asthma, so SFRS8 is a very interesting candidate gene in the region.
Conclusions
Linkage and simulation studies show that the very distal part of chromosome 12q contains a gene that increases the susceptibility to asthma. SFRS8 could act as a weak predisposing gene for asthma in our sample.
doi:10.1136/thx.2005.055475
PMCID: PMC2104763  PMID: 16738036
asthma; genetics; linkage; splicing factor arginine/serine‐rich 8 (SFRS8); atopy
9.  A cAMP-specific phosphodiesterase (PDE8B) that is mutated in adrenal hyperplasia is expressed widely in human and mouse tissues: a novel PDE8B isoform in human adrenal cortex 
Bilateral adrenocortical hyperplasia (BAH) is the second most common cause of corticotropin-independent Cushing syndrome (CS). Genetic forms of BAH have been associated with complex syndromes such as Carney Complex and McCune Albright syndrome or may present as isolated micronodular adrenocortical disease (iMAD) usually in children and young adults with CS. A genome-wide association study identified inactivating phosphodiesterase (PDE) 11A (PDE11A) sequencing defects as low-penetrance predisposing factors for iMAD and related abnormalities; we also described a mutation (c.914A>C/H305P) in cAMP-specific PDE8B, in a patient with iMAD. In this study we further characterize this mutation; we also found a novel PDE8B isoform, highly expressed in the adrenal gland. This mutation is shown to significantly affect the ability of the protein to degrade cAMP in vitro. Tumor tissues from patients with iMAD and no mutations in the coding PDE8B sequence or any other related genes (PRKAR1A, PDE11A) showed down-regulated PDE8B expression (compared to normal adrenal cortex). Pde8b is detectable in the adrenal gland of newborn mice and is widely expressed in other mouse tissues. We conclude that PDE8B is another PDE gene linked to iMAD; it is a candidate causative gene for other adrenocortical lesions linked to the cAMP-signaling pathway, and possibly for tumors in other tissues.
doi:10.1038/ejhg.2008.85
PMCID: PMC2671148  PMID: 18431404
adrenal gland; Cushing syndrome; protein kinase A; cyclic AMP; phosphodiesterases
10.  Phosphodiesterase 4 inhibition enhances the dopamine D1 receptor/PKA/DARPP-32 signaling cascade in frontal cortex 
Psychopharmacology  2011;219(4):1065-1079.
Rationale
Alteration of dopamine neurotransmission in the prefrontal cortex, especially hypofunction of dopamine D1 receptors, contributes to psychotic symptoms and cognitive deficit in schizophrenia. D1 receptors signal through the cAMP/PKA second messenger cascade, which is modulated by phosphodiesterase (PDE) enzymes that hydrolyze and inactivate cyclic nucleotides. Though several PDEs are expressed in cortical neurons, the PDE4 enzyme family (PDE4A-D) has been implicated in the control of cognitive function. The best studied isoform, PDE4B, interacts with a schizophrenia susceptibility factor, disrupted in schizophrenia 1 (DISC1).
Objectives
We explore the control of mouse frontal cortex dopamine D1 receptor signaling and associated behavior by PDE4.
Results
Inhibition of PDE4 by rolipram induced activation of cAMP/PKA signaling in cortical slices and in vivo, leading to the phosphorylation of DARPP-32 and other postsynaptic and presynaptic PKA-substrates. Rolipram also enhanced DARPP-32 phosphorylation invoked by D1 receptor activation. Immunohistochemical studies demonstrated PDE4A, PDE4B and PDE4D expression in DARPP-32-positive neurons in layer VI of frontal cortex, most likely in D1 receptor-positive, glutamatergic corticothalamic pyramidal neurons. Furthermore, the ability of rolipram treatment to improve the performance of mice in a sensorimotor gating test was DARPP-32-dependent.
Conclusions
PDE4, which is co-expressed with DARPP-32 in D1 receptor-positive cortical pyramidal neurons in layer VI, modulates the level of D1 receptor signaling and DARPP-32 phosphorylation in the frontal cortex, likely influencing cognitive function. These biochemical and behavioral actions of PDE4 inhibitors may contribute to the hypothesized antipsychotic actions of this class of compounds.
doi:10.1007/s00213-011-2436-8
PMCID: PMC3539205  PMID: 21833500
PDE4; DARPP-32; PKA; frontal cortex; prepulse inhibition; rolipram
11.  Identification of cytosolic phosphodiesterases in the erythrocyte: A possible role for PDE5 
Medical Science Monitor  2011;17(5):CR241-CR247.
SUMMARY
Background
Within erythrocytes (RBCs), cAMP levels are regulated by phosphodiesterases (PDEs). Increases in cAMP and ATP release associated with activation of β-adrenergic receptors (βARs) and prostacyclin receptors (IPRs) are regulated by PDEs 2, 4 and PDE 3, respectively. Here we establish the presence of cytosolic PDEs in RBCs and determine a role for PDE5 in regulating levels of cGMP.
Methods/Materials
Purified cytosolic proteins were obtained from isolated human RBCs and western analysis was performed using antibodies against PDEs 3A, 4 and 5. Rabbit RBCs were incubated with dbcGMP, a cGMP analog, to determine the effect of cGMP on cAMP levels. To determine if cGMP affects receptor-mediated increases in cAMP, rabbit RBCs were incubated with dbcGMP prior to addition of isoproterenol (ISO), a βAR receptor agonist. To demonstrate that endogenous cGMP produces the same effect, rabbit and human RBCs were incubated with SpNONOate (SpNO), a nitric oxide donor, and YC1, a direct activator of soluble guanylyl cyclase (sGC), in the absence and presence of a selective PDE5 inhibitor, zaprinast (ZAP).
Results
Western analysis identified PDEs 3A, 4D and 5A. dbcGMP produced a concentration dependent increase in cAMP and ISO-induced increases in cAMP were potentiated by dbcGMP. In addition, incubation with YC1 and SpNO in the presence of ZAP potentiated βAR-induced increases in cAMP.
Conclusion
PDEs 2, 3A and 5 are present in the cytosol of human RBCs. PDE5 activity in RBCs regulates cGMP levels. Increases in intracellular cGMP augment cAMP levels. These studies suggest a novel role for PDE5 in erythrocytes.
PMCID: PMC3366467  PMID: 21525805
red blood cell; cGMP; isoproterenol; PDE5; zaprinast
12.  Identification of cytosolic phosphodiesterases in the erythrocyte: A possible role for PDE5 
Summary
Background
Within erythrocytes (RBCs), cAMP levels are regulated by phosphodiesterases (PDEs). Increases in cAMP and ATP release associated with activation of β-adrenergic receptors (βARs) and prostacyclin receptors (IPRs) are regulated by PDEs 2, 4 and PDE 3, respectively. Here we establish the presence of cytosolic PDEs in RBCs and determine a role for PDE5 in regulating levels of cGMP.
Material/Methods
Purified cytosolic proteins were obtained from isolated human RBCs and western analysis was performed using antibodies against PDEs 3A, 4 and 5. Rabbit RBCs were incubated with dbcGMP, a cGMP analog, to determine the effect of cGMP on cAMP levels. To determine if cGMP affects receptor-mediated increases in cAMP, rabbit RBCs were incubated with dbcGMP prior to addition of isoproterenol (ISO), a βAR receptor agonist. To demonstrate that endogenous cGMP produces the same effect, rabbit and human RBCs were incubated with SpNONOate (SpNO), a nitric oxide donor, and YC1, a direct activator of soluble guanylyl cyclase (sGC), in the absence and presence of a selective PDE5 inhibitor, zaprinast (ZAP).
Results
Western analysis identified PDEs 3A, 4D and 5A. dbcGMP produced a concentration dependent increase in cAMP and ISO-induced increases in cAMP were potentiated by dbcGMP. In addition, incubation with YC1 and SpNO in the presence of ZAP potentiated βAR-induced increases in cAMP.
Conclusions
PDEs 2, 3A and 5 are present in the cytosol of human RBCs. PDE5 activity in RBCs regulates cGMP levels. Increases in intracellular cGMP augment cAMP levels. These studies suggest a novel role for PDE5 in erythrocytes.
doi:10.12659/MSM.881763
PMCID: PMC3366467  PMID: 21525805
red blood cell; cGMP; isoproterenol; PDE5; zaprinast
13.  Characterization of Novel Phosphodiesterases in the Bovine Ovarian Follicle1 
Biology of Reproduction  2009;81(2):415-425.
The phosphodiesterase (PDE) family is a group of enzymes that catalyzes the transformation of cyclic nucleotides into 5′ nucleotides. Based on rodents, the current mammalian model of PDE distribution in the ovarian follicle predicts Pde3a in the oocyte and Pde4d in the somatic cells. Using bovine as an experimental model, the present results showed that PDE3 was the predominant PDE activity in oocytes. However, cumulus cell cAMP-PDE activity was predominantly resistant to inhibition by 3-isobutyl-methylxantine, indicating PDE8 activity (60% of total PDE activity) and a minor role for PDE4 (<5%). A total of 20% of total oocyte PDE activity was also attributed to PDE8. The PDE activity measurements in mural granulosa cells from 2 to 6 mm in diameter suggest the presence of PDE4 and PDE8. In granulosa cells from follicles >10 mm, total PDE and PDE8 activities along with PDE8A protein level were increased compared with smaller follicles. The RT-PCR experiments showed that cumulus cells expressed PDE8A, PDE8B, and PDE10A. Western blot experiments showed PDE8A, PDE8B, and PDE4D proteins in mural granulosa cells and cumulus-oocyte complexes. PDE8 inhibition using dipyridamole in a dose-dependent manner increased cAMP levels in the cumulus-oocyte complexes and delayed oocyte nuclear maturation. These results are the first to demonstrate the functional presence of PDE8 in the mammalian ovarian follicle. This challenges the recently described cell-specific expression of cAMP-PDEs in the ovarian follicle and the notion that PDE4 is the predominant granulosa/cumulus cell PDE. These findings have implications for our understanding of hormonal regulation of folliculogenesis and the potential application of PDE inhibitors as novel contraceptives.
PDE8A and PDE8B are the predominant cAMP-phosphodiesterase in bovine cumulus cells and play an important role in cAMP modulation.
doi:10.1095/biolreprod.108.074450
PMCID: PMC2849824  PMID: 19357367
cumulus cells; cyclic adenosine monophosphate; follicle; gamete biology; oocyte; phosphodiesterases; signal transduction
14.  The effect of selective phosphodiesterase inhibitors, alone and in combination, on a murine model of allergic asthma 
Respiratory Research  2004;5(1):4.
Background
The anti-inflammatory effects of the selective phosphodiesterase (PDE) inhibitors cilostazol (PDE 3), RO 20-1724 (PDE 4) and sildenafil (PDE 5) were examined in a murine model of allergic asthma. These compounds were used alone and in combination to determine any potential synergism, with dexamethasone included as a positive control.
Methods
Control and ovalbumin sensitised Balb/C mice were administered orally with each of the possible combinations of drugs at a dose of 3 mg/Kg for 10 days.
Results
When used alone, RO 20-1724 significantly reduced eosinophil influx into lungs and lowered tumour necrosis factor-α, interleukin-4 and interleukin-5 levels in the bronchoalveolar lavage fluid when compared to untreated mice. Treatment with cilostazol or sildenafil did not significantly inhibit any markers of inflammation measured. Combining any of these PDE inhibitors produced no additive or synergistic effects. Indeed, the anti-inflammatory effects of RO 20-1724 were attenuated by co-administration of either cilostazol or sildenafil.
Conclusions
These results suggest that concurrent treatment with a PDE 3 and/or PDE 5 inhibitor will reduce the anti-inflammatory effectiveness of a PDE 4 inhibitor.
doi:10.1186/1465-9921-5-4
PMCID: PMC419478  PMID: 15169552
15.  Mutations in the β-subunit of rod phosphodiesterase identified in consanguineous Pakistani families with autosomal recessive retinitis pigmentosa 
Molecular Vision  2011;17:1373-1380.
Purpose
This study was designed to identify pathogenic mutations causing autosomal recessive retinitis pigmentosa (RP) in consanguineous Pakistani families.
Methods
Two consanguineous families affected with autosomal recessive RP were identified from the Punjab Province of Pakistan. All affected individuals underwent a thorough ophthalmologic examination. Blood samples were collected, and genomic DNAs were extracted. Exclusion analysis was completed, and two-point LOD scores were calculated. Bidirectional sequencing of the β subunit of phosphodiesterase 6 (PDE6β) was completed.
Results
During exclusion analyses both families localized to chromosome 4p, harboring PDE6β, a gene previously associated with autosomal recessive RP. Sequencing of PDE6β identified missense mutations: c.1655G>A (p.R552Q) and c.1160C>T (p.P387L) in families PKRP161 and PKRP183, respectively. Bioinformatic analyses suggested that both mutations are deleterious for the native three-dimensional structure of the PDE6β protein.
Conclusions
These results strongly suggest that mutations in PDE6β are responsible for the disease phenotype in the consanguineous Pakistani families.
PMCID: PMC3108895  PMID: 21655355
16.  Genomewide association study of the age of onset of childhood asthma 
BACKGROUND
Childhood asthma is a complex disease with known heritability and phenotypic diversity. Although an earlier onset has been associated with more severe disease, there has been no genome-wide association study of the age of onset of asthma in children.
OBJECTIVE
To identify genetic variants associated with earlier onset of childhood asthma.
METHODS
We conducted the first genome-wide association study (GWAS) of the age of onset of childhood asthma among participants in the Childhood Asthma Management Program (CAMP), and used three independent cohorts from North America, Costa Rica, and Sweden for replication.
RESULTS
Two SNPs were associated with earlier onset of asthma in the combined analysis of CAMP and the replication cohorts: : rs9815663 (Fisher’s P value=2.31 × 10−8) and rs7927044 (P=6.54 × 10−9). Of these two SNPs, rs9815663 was also significantly associated with earlier asthma onset in an analysis including only the replication cohorts. Ten SNPs in linkage disequilibrium with rs9815663 were also associated with earlier asthma onset (2.24 × 10−7 < P < 8.22 ×10−6). Having ≥1 risk allele of the two SNPs of interest (rs9815663 and rs7927044) was associated with lower lung function and higher asthma medication use during 4 years of follow-up in CAMP.
CONCLUSIONS
We have identified two SNPs associated with earlier onset of childhood asthma in four independent cohorts.
doi:10.1016/j.jaci.2012.03.020
PMCID: PMC3387331  PMID: 22560479
Asthma; pediatrics; age of onset; asthma genetics; C1orf100; genome-wide association study; pediatric asthma
17.  Genome-wide association study of patient and clinician rated global impression severity during antipsychotic treatment 
Pharmacogenetics and genomics  2013;23(2):69-77.
Objective
Examine the unique and congruent findings between multiple raters in a genome-wide association studies (GWAS) in the context of understanding individual differences in treatment response during antipsychotic therapy for schizophrenia.
Methods
We performed GWAS to search for genetic variation affecting treatment response. The analysis sample consisted of 738 patients with schizophrenia, successfully genotyped for ~492K SNPs from the Clinical Antipsychotic Trial of Intervention Effectiveness (CATIE). Outcomes included both clinician and patient report of illness severity on global impression scales, the CGI-S and PGI, respectively. Our criterion for genome-wide significance was a pre-specified threshold ensuring that, on average, only 10% of the significant findings are false discoveries.
Results
Thirteen SNPs reached genome-wide significance. The top findings indicated three SNPs in PDE4D, 5q12.1 (p =4.2×10−8, p =1.6×10−7, p =1.8×10−7), mediated the effects of quetiapine on patient reported severity and an additional three SNPs in TJP1, 15q13.1 (p = 2.25×10−7, p = 4.86×10−7, p = 4.91×10−7), mediated the effects of risperidone on patient reported severity. For clinician reported severity, two SNPs in PPA2, 4q24 (p = 3.68×10−7, p = 5.05×10−7), were found to reach genome-wide significance.
Conclusion
We found evidence of both novel and consistent association when examining the results from the patient and clinician ratings suggesting that different raters may capture unique facets of schizophrenia. Although our findings require replication and functional validation, this study demonstrates the potential of GWAS to discover genes that potentially mediate treatment response of antipsychotic medication.
doi:10.1097/FPC.0b013e32835ca260
PMCID: PMC3922978  PMID: 23241943
CATIE; CGI; pharmacogenomics; personalized medicine; schizophrenia; GWAS; single nucleotide polymorphism; PANSS
18.  β2-Agonist Induced cAMP Is Decreased in Asthmatic Airway Smooth Muscle Due to Increased PDE4D 
PLoS ONE  2011;6(5):e20000.
Background and Objective
Asthma is associated with airway narrowing in response to bronchoconstricting stimuli and increased airway smooth muscle (ASM) mass. In addition, some studies have suggested impaired β-agonist induced ASM relaxation in asthmatics, but the mechanism is not known.
Objective
To characterize the potential defect in β-agonist induced cAMP in ASM derived from asthmatic in comparison to non-asthmatic subjects and to investigate its mechanism.
Methods
We examined β2-adrenergic (β2AR) receptor expression and basal β-agonist and forskolin (direct activator of adenylyl cyclase) stimulated cAMP production in asthmatic cultured ASM (n = 15) and non-asthmatic ASM (n = 22). Based on these results, PDE activity, PDE4D expression and cell proliferation were determined.
Results
In the presence of IBMX, a pan PDE inhibitor, asthmatic ASM had ∼50% lower cAMP production in response to isoproterenol, albuterol, formoterol, and forskolin compared to non-asthmatic ASM. However when PDE4 was specifically inhibited, cAMP production by the agonists and forskolin was normalized in asthmatic ASM. We then measured the amount and activity of PDE4, and found ∼2-fold greater expression and activity in asthmatic ASM compared to non-asthmatic ASM. Furthermore, inhibition of PDE4 reduced asthmatic ASM proliferation but not that of non-asthmatic ASM.
Conclusion
Decreased β-agonist induced cAMP in ASM from asthmatics results from enhanced degradation due to increased PDE4D expression. Clinical manifestations of this dysregulation would be suboptimal β-agonist-mediated bronchodilation and possibly reduced control over increasing ASM mass. These phenotypes appear to be “hard-wired” into ASM from asthmatics, as they do not require an inflammatory environment in culture to be observed.
doi:10.1371/journal.pone.0020000
PMCID: PMC3096656  PMID: 21611147
19.  Phosphodiesterase 3 inhibition and cough in elderly asthmatics 
Aims
Cough is a common symptom of bronchial asthma, a chronic inflammatory airway disease. Recently, the therapeutic effects of selective phosphodiesterase (PDE) inhibitors have been focused on bronchial asthma. This study was designed to investigate the clinical effect of PDE 3 inhibition on cough reflex sensitivity in elderly patients with bronchial asthma.
Methods
Effects of cilostazol, a PDE 3 inhibitor, on cough response to inhaled capsaicin were examined in 11 patients over 70 years with stable asthma in a randomized, placebo-controlled cross over study. Capsaicin cough threshold, defined as the lowest concentration of capsaicin eliciting five or more coughs, was measured as an index of airway cough reflex sensitivity.
Results
The cough threshold was significantly (p < 0.05) increased after two-week treatment with cilostazol (100 mg twice a day orally) compared with placebo [48.8 (GSEM 1.4) vs. 29.2 (GSEM 1.3) μM].
Conclusion
These findings indicate that PDE 3 inhibition may be a novel therapeutic option for elderly patients with asthma, especially for their cough symptoms.
doi:10.1186/1745-9974-1-11
PMCID: PMC1336741  PMID: 16307683
cough reflex sensitivity; capsaicin; cilostazol; phosphodiesterase; bronchial asthma
20.  Phosphodiesterase 4 regulates the migration of B16-F10 melanoma cells 
Phosphodiesterases (PDEs) are important regulators of signal transduction processes. Eleven PDE gene families (PDE1-11) have been identified and several PDE isoforms are selectively expressed in various cell types. PDE4 family members specifically hydrolyze cyclic AMP (cAMP). Four genes (PDE4A-D) are known to encode PDE4 enzymes, with additional diversity generated by the use of alternative mRNA splicing and the use of different promoters. While PDE4 selective inhibitors show therapeutic potential for treating major diseases such as asthma and chronic obstructive pulmonary disease, little is known concerning the role of PDE4 in malignant melanoma. In this study, we examined the role of PDE4 in mouse B16-F10 melanoma cells. In these cells, PDE4 activity was found to be ∼60% of total PDE activity. RT-PCR detected only PDE4B and PDE4D mRNA. Cell growth was inhibited by the cAMP analog, 8-bromo-cAMP, but not by the specific PDE4 inhibitors, rolipram and denbufylline, which increased intracellular cAMP concentrations. Finally, migration of the B16-F10 cells was inhibited by the PDE4 inhibitors and 8-bromo-cAMP, while migration was increased by a protein kinase A (PKA) inhibitor, PKI14–22, and was not affected by 8-pCPT-2′-O-Me-cAMP, which is an analog of exchange protein activated by cAMP (Epac). The inhibitory effect of rolipram on migration was reversed by PKI14–22. Based on these results, PDE4 appears to play an important role in the migration of B16-F10 cells, and therefore may be a novel target for the treatment of malignant melanoma.
doi:10.3892/etm.2012.587
PMCID: PMC3439151  PMID: 22970026
migration; B16-F10 melanoma cells; cyclic AMP; phospho diesterase 4
21.  Genetic associations with neonatal thyroid stimulating hormone levels 
Pediatric research  2013;73(4 0 1):484-491.
Background
Elevations or deficits in thyroid hormone levels are responsible for a wide range of neonatal and adult phenotypes. Several genome-wide, candidate gene and meta-analysis studies have examined thyroid hormones in adults; however, to our knowledge no genetic association studies have been performed with neonatal thyroid levels.
Methods
A population of Iowa neonates; term (n=827) and preterm (n=815), were genotyped for 45 single nucleotide polymorphisms. Thyroid stimulating hormone (TSH) values were obtained from the Iowa Neonatal Metabolic Screening Program. Analysis of variance was performed to identify genetic associations with TSH concentrations.
Results
The strongest association was rs4704397 in the PDE8B gene (p=1.3×10−4), followed by rs965513 (p=6.4×10−4) on chromosome 9 upstream of the FOXE1 gene. Both of these SNPs met statistical significance after correction for multiple testing. Six other SNPs were marginally significant (p<0.05).
Conclusions
We demonstrated for the first time two genetic associations with neonatal TSH levels that replicate findings with adult TSH levels. These SNPs should be considered as early predictors of risk for adult diseases and conditions associated with thyroid hormone levels. Furthermore, this provides a better understanding of the thyroid profile and potential risk for thyroid disorders in newborns.
doi:10.1038/pr.2013.18
PMCID: PMC3775497  PMID: 23344678
22.  Analyses of shared genetic factors between asthma and obesity in children 
Background
Epidemiological studies consistently show associations between asthma and obesity. Shared genetics may account for this association.
Objective
To identify genetic variants associated with both asthma and obesity.
Methods
Based on a literature search, we identified genes from: 1) Genome-wide association studies (GWAS) of Body Mass Index (BMI) (n=17 genes), 2) GWAS of asthma (n=14) and 3) candidate gene studies of BMI and asthma (n=7). We used GWAS data from the Childhood Asthma Management Program (CAMP) to analyze associations between single nucleotide polymorphisms (SNPs) in these genes and asthma (n=359 subjects) and BMI (n=537).
Results
One top BMI GWAS SNP from the literature, rs10938397 near GNPDA2, was associated with both BMI (p=4 × 10−4) and asthma (p=0.03). Of the top asthma GWAS SNPs and the candidate gene SNPs, none was found to be associated with both BMI and asthma. Gene-based analyses that included all available SNPs in each gene found associations (p<0.05) with both phenotypes for several genes: NEGR1, ROBO1, DGKG, FAIM2, FTO and CHST8 among the BMI GWAS genes; ILRL1/IL18R1, DPP10, PDE4D, MYB, PDE10A, IL33 and especially PTPRD among the asthma GWAS genes; and PRKCA among the BMI and asthma candidate genes.
Conclusions
SNPs within several genes showed associations to BMI and asthma at a gene level, but none of these associations were significant after correction for multiple testing. Our analysis of known candidate genes reveals some evidence for shared genetics between asthma and obesity, but other shared genetic determinants are likely to be identified in novel loci.
doi:10.1016/j.jaci.2010.06.030
PMCID: PMC2941152  PMID: 20816195
Association; Asthma; BMI; Children; Genetics; GWAS; Obesity; Polymorphism; SNP
23.  Meta-analysis of genome-wide linkage studies of asthma and related traits 
Respiratory Research  2008;9(1):38.
Background
Asthma and allergy are complex multifactorial disorders, with both genetic and environmental components determining disease expression. The use of molecular genetics holds great promise for the identification of novel drug targets for the treatment of asthma and allergy. Genome-wide linkage studies have identified a number of potential disease susceptibility loci but replication remains inconsistent. The aim of the current study was to complete a meta-analysis of data from genome-wide linkage studies of asthma and related phenotypes and provide inferences about the consistency of results and to identify novel regions for future gene discovery.
Methods
The rank based genome-scan meta-analysis (GSMA) method was used to combine linkage data for asthma and related traits; bronchial hyper-responsiveness (BHR), allergen positive skin prick test (SPT) and total serum Immunoglobulin E (IgE) from nine Caucasian asthma populations.
Results
Significant evidence for susceptibility loci was identified for quantitative traits including; BHR (989 pedigrees, n = 4,294) 2p12-q22.1, 6p22.3-p21.1 and 11q24.1-qter, allergen SPT (1,093 pedigrees, n = 4,746) 3p22.1-q22.1, 17p12-q24.3 and total IgE (729 pedigrees, n = 3,224) 5q11.2-q14.3 and 6pter-p22.3. Analysis of the asthma phenotype (1,267 pedigrees, n = 5,832) did not identify any region showing genome-wide significance.
Conclusion
This study represents the first linkage meta-analysis to determine the relative contribution of chromosomal regions to the risk of developing asthma and atopy. Several significant results were obtained for quantitative traits but not for asthma confirming the increased phenotype and genetic heterogeneity in asthma. These analyses support the contribution of regions that contain previously identified asthma susceptibility genes and provide the first evidence for susceptibility loci on 5q11.2-q14.3 and 11q24.1-qter.
doi:10.1186/1465-9921-9-38
PMCID: PMC2391165  PMID: 18442398
24.  3D-QSAR Study of Potent Inhibitors of Phosphodiesterase-4 Using a CoMFA Approach 
Phosphodiesterase-4 (PDE4) plays an important role in treatment of asthma and chronic obstructive pulmonary disease. Thirty-one analogs displaying variable inhibition of PDE4 were selected to develop models for establishing three-dimensional quantitative structure-activity relationships (3D-QSAR). Comparative molecular field analysis (CoMFA) was conducted on the group of analogs to determine the structural requirements for potency in inhibiting PDE4. The resulting model exhibited good q2 and r2 values up to 0.741 and 0.954 for CoMFA. The contributions from the steric and electrostatic fields were 0.915 and 0.085 respectively. The 3D-QSAR model should be very useful for design of novel PDE 4 inhibitors.
PMCID: PMC3716442
QSAR; CoMFA; Phosphodiesterase-4
25.  Epistatic effects of multiple receptor genes on pathophysiology of asthma – its limits and potential for clinical application 
To date, genome-wide association studies (GWAS) permit a comprehensive scan of the genome in an unbiased manner, with high sensitivity, and thereby have the potential to identify candidate genes for the prevalence or development of multifactorial diseases such as bronchial asthma. However, most studies have only managed to explain a small additional percentage of hereditability estimates, and often fail to show consistent results among studies despite large sample sizes. Epistasis is defined as the interaction between multiple different genes affecting phenotypes. By applying epistatic analysis to clinical genetic research, we can analyze interactions among more than 2 molecules (genes) considering the whole system of the human body, illuminating dynamic molecular mechanisms. An increasing number of genetic studies have investigated epistatic effects on the risk for development of asthma. The present review highlights a concept of epistasis to overcome traditional genetic studies in humans and provides an update of evidence on epistatic effects on asthma. Furthermore, we review concerns regarding recent trends in epistatic analyses from the perspective of clinical physicians. These concerns include biological plausibility of genes identified by computational statistics, and definition of the diagnostic label of ‘physician-diagnosed asthma’. In terms of these issues, further application of epistatic analysis will prompt identification of susceptibility of diseases and lead to the development of a new generation of pharmacological strategies to treat asthma.
doi:10.12659/MSM.889754
PMCID: PMC3907491  PMID: 24435185
clinical application; epistasis; genetic; asthma - physiopathology; disease susceptibility

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