MOLECULAR MEDICINE REPORTS 11: 4463-4467, 2015 Association between the ABCC11 gene polymorphism and the expression of apolipoprotein D by the apocrine glands in axillary osmidrosis ZHECHEN ZHU1, HONGWEI ZHANG1, GUANGHUA LUO2, NING XU3 and ZHONGLAN PAN1 1Department of Plastic and Burn Surgery, The First Affiliated Hospital of Nanjing Medical University, Nanjing, Jiangsu 210003; 2Comprehensive Laboratory, The Third Affiliated Hospital of Soochow University, Changzhou, Jiangsu 213003, P.R. China; 3Section of Clinical Chemistry and Pharmacology, Institute of Laboratory Medicine, Lund University, Lund S-221 85, Sweden Received April 1, 2014; Accepted December 9, 2014 DOI: 10.3892/mmr.2015.3274 Abstract. It has been suggested that the adenosine triphos- gene in the mediation of osmidrosis by enhancing the transi- phate-binding cassette sub-family C member 11 (ABCC11) tion of odor precursors via the ApoD pathway. gene polymorphism and apolipoprotein D (ApoD), an odor precursor carrier, may be important in the formation of axil- Introduction lary odor. To date, few studies have examined the potential correlation between these two factors. The present study Osmidrosis is one of the most common complaints in the aimed to investigate the association between a 538 G>A departments of plastic surgery at the First Affiliated Hospital single-nucleotide polymorphism (SNP) of the ABCC11 gene of Nanjing Medical University (Nanjing, China). The apocrine and the mRNA expression levels of ApoD in the apocrine glands, which are located in the axilla, are the predominant gland of patients with osmidrosis. The 538 G>A polymor- cause of axillary odor (1). This is due to the fat-like secretion phism genotypes of 33 patients with a clinical diagnosis of produced by the apocrine glands, which is broken down into osmidrosis were analyzed by polymerase chain reaction (PCR) volatile odorous substances by bacteria (2). and a base-quenched probe method, and they were divided Certain factors affect and/or contribute to the formation into two groups according to the results. The G allele func- of axillary odor, including genetics, gender, age, develop- tions as a dominant gene; therefore, patients with the GG or mental stage, diet, treatment history and family history (3-5). GA genotype were allocated to Group I (n=28) and patients However, the mechanisms underlying the development with the AA genotype to Group II (n=5). The mRNA expres- of osmidrosis remain to be elucidated. A previous study sion levels of ApoD in the apocrine glands were determined demonstrated that genetic and environmental factors are by reverse transcription-PCR. The results indicated that the considered the most significant factors in the development of mRNA expression levels of ApoD were significantly higher in osmidrosis (6). The effect of genetics as a vital contributing the apocrine glands of patients in Group I compared with those factor is also of interest. Increasing evidence has indicated that in Group II (P<0.01). In conclusion, the results indicated that the single nucleotide polymorphism (SNP), rs17822931 (538 the ABCC11 gene SNP of the 538 G>A allele was associated G>A), of the ABCC11 gene located on human chromosome with a downregulation of the mRNA expression of ApoD in 16q12.1 is associated with axillary osmidrosis (3). ABCC11 the apocrine glands, which may indicate a role for the ABCC11 is expressed and localized in the apocrine glands and has a key function in the secretion of odorants and their precur- sors (7). A previous study revealed that ~98.7% of individuals with osmidrosis have the GG or GA genotype (7), which is a significantly larger proportion compared with the overall population and suggests that the G allele of ABCC11 may be Correspondence to: Professor Hongwei Zhang, Department of important in the expression of axillary odor. Apolipoprotein D Plastic and Burn Surgery, The First Affiliated Hospital of Nanjing (ApoD), a 29 kDa glycoprotein, is the primary protein compo- Medical University, 1 Zhongfu Road, Nanjing, Jiangsu 210003, P. R. Ch i na nent of high-density lipoprotein in human plasma (8). It has E-mail: [email protected] been demonstrated that ApoD is a physiological carrier of odor precursors in vivo and its sequence is also expressed Key words: osmidrosis, adenosine triphosphate-binding cassette in the apocrine glands (9), indicating a role for ApoD in the sub-family C member 11, single-nucleotide polymorphism, transition of axillary odor precursors. Furthermore, it has been apolipoprotein D revealed that levels of ApoD are increased in individuals with osmidrosis (4). Associations between the rs17822931 SNP of ABCC11, ApoD and axillary odor have been observed. 4464 ZHU et al: ASSOCIATION BETWEEN THE ABCC11 POLYMORPHISM AND ApoD EXPRESSION IN OSMIDROSIS Notably, the ethnic distribution of the ABCC11 G allele is Table I. Stages of odor severity. similar to that of high-level apocrine ApoD (10). To date, few studies have investigated the correlation between ABCC11 and Stage Distance from odor detection (cm) ApoD. Therefore, the present study aimed to further examine the association between the ABCC11 genotype at rs17822931 One <15 and the mRNA expression levels of ApoD in the apocrine Two ≥15 and <30 glands of patients with osmidrosis. Three ≥30 and <100 Four ≥100 Materials and methods Patients and samples. All procedures were performed according to protocols approved by the Ethical Review Board template, 2.5 µl 10X PCR buffer, 1.5 µl 25 mM MgCl2, 0.5 µl of the First Affiliated Hospital of Nanjing Medical University 4X dNTPs, 1.25 U Taq DNA polymerase, 0.1 µl 100 µM of each (Nanjing, China) between November 2012 and August 2013. primer and 0.2 µl 10 µM probes in a final reaction volume of A total of 33 patients exhibiting symptoms of axillary odor 25 µl. Thermal cycling for ABCC11 (538G>A) was performed and receiving surgery in the Department of Plastic and on a LightCycler (Version 480II; Roche Diagnostics, Basel, Burn Surgery (First Affiliated Hospital of Nanjing Medical Switzerland) under the following conditions: 5 min of initial University) were included in the study following the provi- denaturation at 95˚C, followed by 40 cycles at 95˚C for 1 sec sion of written informed consent. Peripheral blood (2 ml) (temperature transition rate 4.4˚C/sec), 62˚C for 25 sec and 72˚C was drawn from all subjects using into EDTA-containing for 10 sec. The analytical melting program involved melting the tubes (BD vacutainer; BD Biosciences, Franklin Lakes, NJ, PCR products at 95˚C for 1 min, 35˚C for 2 min and increasing USA) and stored at ‑20˚C to obtain genomic DNA. The gender, the temperature to 70˚C at a transition rate of 0.06˚C/sec, age, height, weight, dietary preferences, treatment history and with continuous acquisition of fluorescence data using the family history of each patient were recorded. To evaluate the LightCycler, as previously described (11). The sequences of the odor severity, the patients were required to bathe 1 day prior homozygous G genotype and the base-quenched probe formed to their procedure and rest in an examination room (22˚C) for an exact match resulting in a higher melting temperature (TM) 30 min prior to surgery. The entire axilla was then exposed to enable the different genotypes to be distinguished (12). for odor evaluation, which was performed by a researcher and Subsequently, seven samples were randomly selected and plastic surgeon. Four distinct stages were outlined according sequenced on an automatic sequencer (Model 3730, Applied to the distance from which the odor was sensed, which are Biosystems, Invitrogen Life Technologies, Shanghai, China) to outlined in Table I. When there was a disagreement in stage verify the genotyping results. designation, the stage was evaluated by an additional researcher and the median was used. All the patients received surgery Reverse transcription‑PCR analyses of the mRNA expres‑ involving micro-incision subcutaneous trimming under local sion of ApoD. The total RNA of apocrine gland tissues were anesthesia (Lidocaine, 2% diluted with saline to 1%; Shanghai extracted using the Total RNAPurification kit (Sangon Biotech Fuxing Chaohui Pharmaceutical Co., Ltd., Shanghai, China). Co., Ltd, Shanghai, China), according to the manufacturer's The subcutaneous tissues, including the apocrine glands of the instructions, no prior steps were required. The quality of the axilla, were removed during surgery, immediately frozen in RNA samples was evaluated by measuring the absorbance at liquid nitrogen (Changzhou Changyu Practical Gas Co., Ltd., 260/280 nm (Biophotometer; Eppendorf, Hamburg, Germany). Changzhou, China) and transferred for storage at -80˚C. Total RNA (2 µg) was reverse transcribed to cDNA using a RevertAid First‑Strand cDNA Synthetic kit (Thermo Scientific, Base‑quenched probe genotyping of the ABCC11 (538 G>A) Waltham, MA, USA) according to the manufacturer's instruc- polymorphism. Individual genomic DNA was extracted from tions. Sequence data for human ApoD mRNA was obtained 250 µl samples of peripheral blood using a 3S Blood DNA from the NCBI database. Primer Premier 5.0 software was Isolation kit (Shenergy Biocolor Co., Shanghai, China), used to design the primers and probes for the human ApoD and according to the manufacturer's instructions. Sequence GAPDH genes. The primers and probes are shown in Table III. data for the human ABCC11 gene was obtained from the GAPDH was used as the reference gene. Amplifications National Center for Biotechnology Information (NCBI) data- were performed in a 7300 Real-time PCR system (Applied base (http://www.ncbi.nlm.nih.gov/gene/85320#reference-se Biosystems Life Technologies, Foster City, CA, USA). The quences). Primer Premier 5.0 (Premier Biosoft International, optimum reaction conditions were obtained using 2.5 µl 10X Palo Alto, CA, USA) was used to design a pair of primers and a PCR buffer, 2.5 µl 25 mm MgCl2, 0.5 µl 10 mm 4X dNTPs, probe to distinguish the nucleotide (538 G>A).