RESUMO
BACKGROUND: Aspirin-exacerbated respiratory disease (AERD) is characterized by the triad of chronic rhinosinusitis with nasal polyps (CRSwNP), asthma, and intolerance to cyclooxygenase-1 enzyme inhibitors. The underlying mechanisms contributing to AERD pathogenesis are not fully understood, but AERD is characterized by an enhanced type 2 inflammatory phenotype. Basophils are potent type 2 effector cells, but their involvement in AERD pathophysiology remains unclear. OBJECTIVE: We sought to characterize the systemic and local basophil responses in patients with AERD compared with patients with CRSwNP. METHODS: Sinonasal tissues including inferior turbinate and/or nasal polyps (NPs) and peripheral blood were collected from controls, patients with AERD, and patients with CRSwNP. Expression of cell surface (CD45, FcεRI, CD203c), activation (CD63), and intracellular (2D7) markers associated with basophils was characterized using flow cytometry. Clinical data including Lund-Mackay scores and pulmonary function were obtained. RESULTS: The mean number of basophils (CD45+CD203c+FcεRI+CD117-) detected in AERD NPs (147 ± 28 cells/mg tissue) was significantly elevated compared with that detected in CRSwNP NPs (69 ± 20 cells/mg tissue; P = .01). The number of circulating basophils was significantly elevated in patients with AERD (P = .04). Basophils in NPs had significantly higher CD203c and CD63 mean fluorescence intensity compared with blood in both conditions (P < .01). Basophils from AERD NPs had lower expression of the granule content marker 2D7 compared with those from matched blood (P < .01) or NPs of patients with CRSwNP (P = .06), suggesting ongoing degranulation. Basophil 2D7 mean fluorescence intensity significantly correlated with pulmonary function (r = 0.62; P = .02) and inversely correlated with sinonasal inflammation (r = -0.56; P = .004). CONCLUSIONS: Increased basophil numbers and extent of ongoing degranulation in NPs of patients with AERD compared with patients with CRSwNP may contribute to the exaggerated disease pathogenesis and severity unique to AERD.
Assuntos
Asma/imunologia , Basófilos/imunologia , Inibidores de Ciclo-Oxigenase/efeitos adversos , Pólipos Nasais/imunologia , Rinite/imunologia , Sinusite/imunologia , Adulto , Asma/induzido quimicamente , Asma/patologia , Basófilos/patologia , Doença Crônica , Inibidores de Ciclo-Oxigenase/uso terapêutico , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Pólipos Nasais/induzido quimicamente , Pólipos Nasais/patologia , Rinite/induzido quimicamente , Rinite/patologia , Sinusite/induzido quimicamente , Sinusite/patologiaRESUMO
One of the causes of coronary heart disease (CHD) is genetic factors. In this study, we explored the relationship between CYP2D7 and TCF20 gene polymorphisms and the risk of CHD in the Han Chinese population. Three single nucleotide polymorphisms (CYP2D7 rs1800754, CYP2D7 rs2743461, and TCF20 rs760648) were selected and genotyped from 490 cases and 480 controls. The odds ratios (ORs) and 95% confidence intervals (CIs) were used to assess the association between CYP2D7 and TCF20 polymorphisms and the risk of CHD. The association between clinical indicators and polymorphisms was analyzed using one-way ANOVA and Tukey's HSD. The SNP-SNP interactions were obtained by performing multifactor dimensionality reduction (MDR). CYP2D7 rs1800754 and rs2743461 were closely associated with increased risk of CHD (alleles: p = 0.014, p = 0.031). Stratified analysis showed that CYP2D7 rs1800754 and rs2743461 were associated with an increased risk of CHD in men, age > 60 years, BMI ≥ 24, and smoking. Rs1800754 is also associated with an increased risk of CHD associated with alcohol consumption. In addition, TCF20 rs760648 was associated with a reduced risk of CHD in patients aged ≤ 60 years and with CALs. A significant association was found between CYP2D7 rs1800754 and rs2743461 genotypes and levels of UREA, Cr, and LDL-C; TCF20 rs760648 genotypes and levels of RBC. The MDR analysis showed that the three-locus interaction model was the best in the multi-locus model. In conclusion, CYP2D7 rs1800754 and rs2743461 polymorphisms were associated with CHD risk.
RESUMO
Genotyping of the CYP2D6 gene is the most commonly applied pharmacogenetic test globally. Significant economic interests have led to the development of a plurality of assays, available for almost any genotyping platform or DNA detection chemistry. Of all the genetic variants, copy number variations are particular difficult to detect by polymerase chain reaction. Here, we present two simple novel approaches for the identification of samples carrying either deletions or duplications of the CYP2D6 gene; by relative quantification using a singleplex 5'nuclease real-time PCR assay, and by high-resolution melting of PCR products. These methods make use of universal primers, targeting both the CYP2D6 and the reference gene CYP2D8P, which is necessary for the analysis. The assays were validated against a reference method using a large set of samples. The singleplex nature of the 5'nuclease real-time PCR ensures that the primers anneal with equal affinity to both the sequence of the CYP2D6 and the reference gene. This facilitates robust identification of gene deletions and duplications based on the cycle threshold value. In contrast, the high-resolution melting assay is an end-point PCR, where the identification relies on variations between the amount of product generated from each of the two genes.
RESUMO
The ultrarapid CYP2D6 metabolizer (UM) phenotype is caused by CYP2D6 gene duplications in some, but not all, UM individuals. CYP2D6 and the adjacent pseudogene CYP2D7 are highly homologous; however, CYP2D7 harbors a premature stop codon, which is absent in carriers of the rare CYP2D7 variant rs530303678. We addressed whether rs530303678 could generate a functionally active protein, causing the UM phenotype. However, unlike CYP2D6 variants, two CYP2D7 rs530303678 variant isoforms, previously described in liver, showed neither significant protein expression nor catalytic activity toward the CYP2D6 substrates bufuralol or dextromethorphan. We conclude that loss of the stop codon in CYP2D7 does not result in the generation of enzymatically active protein in human liver and thus, cannot cause the UM phenotype.
Assuntos
Sistema Enzimático do Citocromo P-450/genética , Variação Genética/genética , Sequência de Aminoácidos , Linhagem Celular , Humanos , Fígado/enzimologiaRESUMO
OBJECTIVE Our understanding of pediatric cervical spine development remains incomplete. The purpose of this analysis was to quantitatively define cervical spine growth in a population of children with normal CT scans. METHODS A total of 1458 children older than 1 year and younger than 18 years of age who had undergone a cervical spine CT scan at the authors' institution were identified. Subjects were separated by sex and age (in years) into 34 groups. Following this assignment, subjects within each group were randomly selected for inclusion until a target of 15 subjects in each group had been measured. Linear measurements were performed on the midsagittal image of the cervical spine. Twenty-three unique measurements were obtained for each subject. RESULTS Data showed that normal vertical growth of the pediatric cervical spine continues up to 18 years of age in boys and 14 years of age in girls. Approximately 75% of the vertical growth occurs throughout the subaxial spine and 25% occurs across the craniovertebral region. The C-2 body is the largest single-segment contributor to vertical growth, but the subaxial vertebral bodies and disc spaces also contribute. Overall vertical growth of the cervical spine throughout childhood is dependent on individual vertebral body growth as well as vertical growth of the disc spaces. The majority of spinal canal diameter growth occurs by 4 years of age. CONCLUSIONS The authors' morphometric analyses establish parameters for normal pediatric cervical spine growth up to 18 years of age. These data should be considered when evaluating children for potential surgical intervention and provide a basis of comparison for studies investigating the effects of cervical spine instrumentation and fusion on subsequent growth.
Assuntos
Vértebras Cervicais/diagnóstico por imagem , Vértebras Cervicais/crescimento & desenvolvimento , Tomografia Computadorizada por Raios X , Adolescente , Criança , Desenvolvimento Infantil , Pré-Escolar , Feminino , Humanos , Lactente , Masculino , Caracteres SexuaisRESUMO
TaqMan™ genotyping assays are widely used to genotype CYP2D6, which encodes a major drug metabolizing enzyme. Assay design for CYP2D6 can be challenging owing to the presence of two pseudogenes, CYP2D7 and CYP2D8, structural and copy number variation and numerous single nucleotide polymorphisms (SNPs) some of which reflect the wild-type sequence of the CYP2D7 pseudogene. The aim of this study was to identify the mechanism causing false-positive CYP2D6 (*) 15 calls and remediate those by redesigning and validating alternative TaqMan genotype assays. Among 13,866 DNA samples genotyped by the CompanionDx® lab on the OpenArray platform, 70 samples were identified as heterozygotes for 137Tins, the key SNP of CYP2D6 (*) 15. However, only 15 samples were confirmed when tested with the Luminex xTAG CYP2D6 Kit and sequencing of CYP2D6-specific long range (XL)-PCR products. Genotype and gene resequencing of CYP2D6 and CYP2D7-specific XL-PCR products revealed a CC>GT dinucleotide SNP in exon 1 of CYP2D7 that reverts the sequence to CYP2D6 and allows a TaqMan assay PCR primer to bind. Because CYP2D7 also carries a Tins, a false-positive mutation signal is generated. This CYP2D7 SNP was also responsible for generating false-positive signals for rs769258 (CYP2D6 (*) 35) which is also located in exon 1. Although alternative CYP2D6 (*) 15 and (*) 35 assays resolved the issue, we discovered a novel CYP2D6 (*) 15 subvariant in one sample that carries additional SNPs preventing detection with the alternate assay. The frequency of CYP2D6 (*) 15 was 0.1% in this ethnically diverse U.S. population sample. In addition, we also discovered linkage between the CYP2D7 CC>GT dinucleotide SNP and the 77G>A (rs28371696) SNP of CYP2D6 (*) 43. The frequency of this tentatively functional allele was 0.2%. Taken together, these findings emphasize that regardless of how careful genotyping assays are designed and evaluated before being commercially marketed, rare or unknown SNPs underneath primer and/or probe regions can impact the performance of PCR-based genotype assays, including TaqMan. Regardless of the test platform used, it is prudent to confirm rare allele calls by an independent method.