The Role of Collagen VI α6 Chain Gene in Atopic Dermatitis

Background@#In a previous study, we carried out whole-exome sequencing to identify genetic variants associated with early onset atopic dermatitis (AD) in Koreans and found that collagen VI α6 chain (COL6A6) gene polymorphisms are associated. COL6A6 is one of the chains that makes up the triple helix of collagen VI, and little is known about its role in AD. @*Objective@#To identify how COL6A6 changes in AD and clarify its role. @*Methods@#Immunohistochemical staining for COL6A6 was performed on tissues of AD, other skin diseases, and healthy controls. Human keratinocytes and fibroblasts were exposed to inflammatory cytokines and cultured to evaluate changes in COL6A6 expression. COL6A6 small interfering RNA (siRNA) was transfected into cells to identify the role of COL6A6. @*Results@#Total COL6A6 mRNA was higher in AD than in controls. In AD tissues, COL6A6 mRNA decreased significantly in the epidermis compared to controls, whereas COL6A6 protein was increased in the dermis. In the cultured cells, COL6A6 mRNA was suppressed in the epidermis by interleukin (IL)-4 and IL-13, whereas COL6A6 protein was induced in the dermis. In the COL6A6 siRNA-transfected keratinocyte, mRNA of FLG, LOR, and CASP14 decreased compared to controls; in contrast, mRNA of MMP1 increased. @*Conclusion@#The reduction of epidermal COL6A6 due to the genetic mutation can cause skin barrier damage and it can contributes to the early onset of AD. COL6A6 is induced by IL-4 and IL-13, and it may play a role in fibrotic remodeling and inflammatory processes, which are major features of AD.

Association of DOCK8, IL17RA, and KLK12 Polymorphisms with Atopic Dermatitis in Koreans

Background@#Early-onset and severe atopic dermatitis (AD) in patients increase the probability of the development of allergic rhinitis or asthma. Treatment and prevention strategies in infants and young children with AD are targeted toward treating the symptoms, restoring skin barrier functions, and reducing the absorption of environmental allergens in an attempt to attenuate or block the onset of asthma and food allergy. @*Objective@#Given that the initiating events in AD remain poorly understood, identifying those at risk and implementing strategies to prevent AD is necessary. @*Methods@#Whole-exome sequencing (WES) was performed in a 43 control group and a disease group with 20 AD patients without atopic march (AM) and 20 with AM. Sanger sequencing was carried out to validate found variants in cohorts. @*Results@#DOCK8, IL17RA, and KLK12 single-nucleotide polymorphisms were identified by WES as missense mutations: c.1289C＞ A, p.P97T (rs529208); c.1685C＞A, p.P562G (rs12484684); and c.457+27＞C, rs3745540, respectively. A case-control study show that total immunoglobulin E (IgE) level was significantly increased in the AA genotype of DOCK8 compared to the CA genotype in allergic patients. The rs12484684 of IL17RA increased risk of adult-onset AD (odds ratio: 1.63) compared to the control for (A) allele frequency. AD and AM Patients with the IL17RA CA genotype also had elevated IgE levels. rs3745540 of KLK12 was associated with AD in dominant model (odds ratio: 2.86). @*Conclusion@#DOCK8 (rs529208), IL17RA (rs12484684), and KLK12 (rs3745540), were identified using a new WES filtering method. the result suggests that polymorphism of DOCK8 and IL17RA might be related to increase the total IgE level.

TSLP Polymorphisms in Atopic Dermatitis and Atopic March in Koreans

BACKGROUND: Atopic march (AM) is the progression from atopic dermatitis (AD) to allergic rhinitis and asthma. The development of AD is as high as 20% in children worldwide and continues to increase. AD seems to be caused by both genetic and environmental factors. Recently, polymorphisms of the thymic stromal lymphopoietin (TSLP) gene associated with allergic disorders were reported in ethnic groups from various countries. OBJECTIVE: Identification of TSLP polymorphisms in Koreans with AD or AM. METHODS: Whole-exome sequencing was performed in 20 AD and 20 AM patients. RESULTS: Nine single nucleotide polymorphisms (SNPs) of TSLP were detected (rs191607411, rs3806933, rs2289276, rs2289277, rs2289278, rs139817258, rs11466749, rs11466750, rs10073816). These SNPs have been correlated with susceptibility to allergic diseases in ethnic groups from China, Japan, Turkey, and Costa Rica in previous studies. Remarkably, one of 20 patients in the AD group lacked all SNPs, compared to six of 20 patients in the AM group. Odds ratios showed that Korean patients without the nine TSLP variants had an 8.14 times higher risk of moving from AD to AM. Two haplotype blocks were validated in 60 AD and 59 AM patients using Sanger sequencing. The haplotype blocks (rs3806933 and rs2289276) and (rs11466749 and rs11466750) were in high linkage disequilibrium, respectively (D′=0.97, D′=1). CONCLUSION: The increase of major allele frequency of respective nine TSLP variants may enhance the risk of AM. These data will contribute to improved genetic surveillance system in the early diagnosis technology of allergic disease.

Erratum: Association of CDKAL1 Polymorphisms with Early-Onset Atopic Dermatitis in Koreans

The original version of the article contained a mistake. The authors have attached a corrected version of Table 3.

Association of CDKAL1 Polymorphisms with Early-Onset Atopic Dermatitis in Koreans

BACKGROUND: Atopic dermatitis (AD) has increased in frequency to rates as high as 20% for children in developed countries. AD is one of the most common childhood diseases and has a complex etiology involving genetic and environmental factors. Thus, a broad understanding of genetic background is needed for early diagnosis of AD. OBJECTIVE: Identification of candidate functional genetic variants associated with early-onset AD in Koreans. METHODS: Whole-exome sequencing (WES) was performed in three families. Sanger sequencing was used to validate detected variants in 112 AD patients and 61 controls. RESULTS: Functional variants were filtered by WES, and then variants related to allergic immune diseases were selected through a literature search. Two candidate non-synonymous single-nucleotide polymorphisms of CDKAL1 (rs77152992) and ERBB2 (rs1058808) were identified, c.1226C>T, p.Pro409Leu, c.3463C>G, and p. Pro1170Ala respectively. A case-control study was performed to determine whether rs77152992 and rs1058808 are candidate risk factors for early-onset AD. rs77152992 was significantly associated with early-onset AD (odds ratio [OR], 0.42; 95% confidence interval [CI], 0.21~0.83; p=0.0133) in allele frequencies. The CC genotype of CDKAL1 had significantly increased risk of AD (OR, 2.16; 95% CI, 1.0~4.6; p=0.0475). rs1058808 had no correlation with AD. Total eosinophil count was significantly increased in AD patients with the CC genotype of CDKAL1 (rs77152992). CONCLUSION: CDKAL1 (rs77152992) and ERBB2 (rs1058808) were deemed functionally interesting based on WES. Our case-control study suggests that the CC genotype of rs77152992 may be associated with increased eosinophil counts. It may enhance the risk of early-onset AD.

Production of Egg Yolk Antibodies Specific to House Dust Mite Proteins

PURPOSE: House dust mites (HDMs) are an important source of indoor allergens associated with asthma, rhinitis and atopic dermatitis. Chicken immunoglobulin (Ig) Y is known to be a good alternative to mice and rabbit antibody production. In this study, we produced IgYs specific to HDMs and investigated their IgE immunoreactivities. MATERIALS AND METHODS: Total IgYs were isolated from the yolks of White Leghorn hens immunized with either Dermatophagoides pteronyssinus or D. farinae protein extract. Control antibodies were separated from the yolks of immunized hens with phosphate buffered saline. IgYs specific to HDMs were analyzed using enzyme-linked immunosorbent assay and Western blotting analysis. RESULTS: The concentration of egg IgY specific to D. farinae in an immunized hen increased and the highest achieved was 661.3 ug/mg (per an egg) on day 47, compared with 760 ug/mg IgY specific to D. pteronyssinus on day 16. The D. pteronyssinus or D. farinae-specific IgY was detected by binding of each mite proteins, and their immunoreactivities were elevated dependent of the specific IgY concentration. CONCLUSION: IgY specific to HDMs may be a promising antibody for immunological diagnosis as well as identification of possible resistance relating to HDM allergy.

The effect of heat treatment or hydrolysis on cow's milk protein distributions and antigenicities

PURPOSE: Cow's milk protein is one of the most common and strongest food allergen. We investigated the effects of heat treatment on the distribution and antigenicities of major allergens from cow's milk. We also compared the protein distribution and antigenicities among cow's milk formula and its substitutes. METHODS: We heated alpha-casen, beta-lactoglobulin (BLG), alpha-lactalbumin (ALA), and crude extract of cow's milk in 100degrees C boiling water for 1 hour. We prepared crude extracts from cow's milk formula, partially hydrolyzed milk formula (pHF) and extensively hydrolyzed milk formula (eHF). The protein compositions of all the samples were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The antigenicities were determined by IgE immunoblotting with pooled serum collected from 11 patients with milk allergy. RESULTS: After heating, no significant alteration was found in casein, and the aggregates of ALA and BLG were detected with molecular weights of about 30 and 45 kDa, respectively. The antigenicities of newly detected aggregates were increased. The new aggregates of BLG with increased antigenicities were also found in heated milk total protein. Major milk allergens were not found in pHF, and residual components with a molecular weight below 10 KDa did not show IgE-binding activity. We failed to observe the residual components and antigenicities of eHF. CONCLUSION: Changes in protein distribution and antigenicity of milk total protein induced by heat treatment may not be significantly different from those of each major allergen. The residual components of pHF could have little IgE-binding capacity, and there may be few or no antigenic components in eHF.

Increased inflammatory mediator in exhaled breath condensate from asthmatic children

PURPOSE: There has recently been increasing interest in the use of exhaled breath condensate (EBC) as a simple noninvasive means for understanding the physiology of asthma. The aim of this study was to evaluate the levels of leukotriene B4 (LTB4) and eosinophil cationic protein (ECP) in the EBC of asthmatic children. METHODS: We measured LTB4 and ECP levels in EBC from children aged 6-14 years, including healthy children (n=25) and asthmatic children (n=25). We also measured serum LTB4 and serum ECP. Pulmonary function tests and methacholine challenge tests were performed on all subjects. RESULTS: Exhaled LTB4 levels were increased significantly in patients with asthma compared to normal subjects (7.1+/-3.7 pg/mL vs. 2.2+/-1.7 pg/mL, P<0.05). Serum LTB4 levels were not significantly different in patients with asthma compared to normal subjects (674.7+/-484.1 pg/mL vs. 487.1+/-272.0 pg/mL, P=0.156,) and no significant correlations were found between exhaled and serum LTB4 concentrations in children with asthma (r=0.052, P=0.758). Exhaled ECP levels were not significantly different in patients with asthma compared to normal subjects (P=0.419). Serum ECP levels were significantly increased in patients with asthma compared to normal subjects (44.37+/-32.14 microg/L vs. 16.40+/-13.23 microg/L, P=0.001). CONCLUSION: We found significantly elevated LTB4 levels in the EBC of asthmatic children. Our results suggest that EBC may be one of the supportive tools to measure airway inflammation in children with asthma.

Erratum: Involvement of Human Histamine N-Methyltransferase Gene Polymorphisms in Susceptibility to Atopic Dermatitis in Korean Children

We found an error in our published article, Table 3.

Involvement of Human Histamine N-Methyltransferase Gene Polymorphisms in Susceptibility to Atopic Dermatitis in Korean Children

PURPOSE: Histamine N-methyltransferase (HNMT) catalyzes one of two major histamine metabolic pathways. Histamine is a mediator of pruritus in atopic dermatitis (AD). The aim of this study was to evaluate the association between HNMT polymorphisms and AD in children. METHODS: We genotyped 763 Korean children for allelic determinants at four polymorphic sites in the HNMT gene: -465T>C, -413C>T, 314C>T, and 939A>G. Genotyping was performed using a TaqMan fluorogenic 5' nuclease assay. The functional effect of the 939A>G polymorphism was analyzed. RESULTS: Of the 763 children, 520 had eczema and 542 had atopy. Distributions of the genotype and allele frequencies of the HNMT 314C>T polymorphism were significantly associated with non-atopic eczema (P=0.004), and those of HNMT 939A>G were significantly associated with eczema in the atopy groups (P=0.048). Frequency distributions of HNMT -465T>C and -413C>T were not associated with eczema. Subjects who were AA homozygous or AG heterozygous for 939A>G showed significantly higher immunoglobulin E levels than subjects who were GG homozygous (P=0.009). In U937 cells, the variant genotype reporter construct had significantly higher mRNA stability (P<0.001) and HNMT enzyme activity (P<0.001) than the common genotype. CONCLUSIONS: Polymorphisms in HNMT appear to confer susceptibility to AD in Korean children.