The Study On Axillary Osmidrosis Genetic Pathogenesis And Its Surgical Treatment

PartⅠ The study on osmidrosis genetic pathogenesis:The relationship between a functional ABCC11 allele and biochemical formation of osmidrosis in ChineseIntroductionMany people nowadays worry about their axillary odour and use deodorants and antiperspirants. The annual sales of these commercial products amount to over $1.9 billion in the United States. In the United Kingdom, more than 90% of women use deodorants or antiperspirants on a daily basis, while 80% of men do so. In Asian countries, the number of osmidrosis is less than 5%, but still costs a lot on treatment because of cultural values. As the development in pathogenesis, four ways of odorants formation by the action of aerobic corynebacteria on odorant precursors were identified.ATP-binding cassette (ABC) proteins form one of the largest protein families encoded in the human genome are involved in membrane transport of drugs, xenobiotics, endogenous substances, or ions, thereby exhibiting a wide spectrum of biological functions. In 2001, three research groups independently cloned two novel ABC transporters named ABCC11 and ABCC12 from the Cdna library of human adult liver. ABCC11 gene that encodes an apical efflux pump was found to be able to transport some of the precursors and is crucial for the formation of the characteristic ear wax and axillary odor. Individuals who were homozygotic for single-nucleotide polymorphism (SNP) (538G>A) (in ABCC11 gene) that changes amino acid 180 in the resultant protein’s polypeptide chain from glycine (G) to arginine (R) were found to lost characteristic axillary odorants. The SNP 538G>A, which is prominent among Asian people, leads to a nearly complete loss of the typical odor components in axillary sweat. The secretion of amino-acid conjugates of human-specific odorants is abolished in homozygotic carriers of the SNP, and steroidal odorants and their putative precursors are significantly reduced.The mechanism of generating these precursors is still not clear. How ABCC11 play role in the formation of body odour does is unknown. In this study, we sequence part of the ABCC11 gene to find the relationship between SNP 538G>A and biochemical formation of osmidrosis in Chinese patients.Materials and MethodsTissue samplesAxillary tissue samples were collected from experiment group (n=12). All members in experiment were dignosed with osmidrosis. Among them, six accepted axillary apocrine suction-curettage surgical treatment and the other six received traditional axillary apocrine surgical way. The control sample is aquired from control group that made of two patients, who were dignosed with breast cancer and needed radical operation of mastocarcinoma. All the tissue samples (n=14) were from axillary subcutaneous. Allvolunteers were informed about the aim and purpose of this study and were asked to give written consent.Histology AssayMorphologic criteria of apocrine were evaluated in serial HE-stained sections under light microscopy.ABCC11 genotypingGenomic DNA was purified from the axillary samples according to the manufacturer’s instructions. PCR amplification was carried out using primers ABCC11 for (5’-CTCCCACATCCTCAATTCTCTGC-3’) and ABCC11_rev (5’-GCCATCCATCGTGTGGAAGAT -3’)-The amplified 354-bp fragment was purified using the ABI PRISM 7500 RT-PCR System and was subsequently sequenced using primer ABCC11 for. The ABCC11 genotype (SNP at 538, G-A) of 14 samples was analyzed.RT-PCRTotal apocrine mRNA was isolated from small pieces of axillary tissue by using the TRIzol reagent. Reverse transcription was performed on 3 μg of RNA with an oligo (dT) primer. SYBR Green was used for the quantification of PCR reactions.The prepared cDNA was subjected to PCR in the presence of primers. ABCC11 for (5’-CTCCCACATCCTCAATTCTCTGC-3’) and ABCC11_rev (5’-GCCATCCATCGTGTGGAAGAT-3’) were used. Human GAPDH/PolR2A were used as double endogenous controls to standardize the amount of cDNA added to the reaction. The PCR amplification was placed in an ABI PRISM 7500 RT-PCR System.The PCR reaction consisted of the following steps:1) initial 3 min denaturation at 94℃; 2) 40 cycles of denaturation at 94℃ for 20 sec and annealing extension at 60℃ for 45 sec. ΔCt value was defined as the absolute value of the difference between the Ct value of the target gene and GAPDH/PolR2A for each sample.StatisticsAll data are presented as mean values ±standard error of the mean (SEM). Wilcoxon test of independent means were used for statistical analysis. Data were considered significant at a level of P<0.05.RESULTSMacroscopic FindingsIn the experiment group, the tissue samples were small pieces, like a mixture of fresh yellow fat tissue and hair follicles were seen. In the control group, each sample was a piece of fat tissue, seldom seen hair follicles.Histology AssayAll the samples were HE-stained, and sections were evaluated under light microscopy. Lumens of apocrine sweat gland and secretory cells were seen in HE-stained sections of both experiment and control groups. But the secretory cells are morphological different in two groups.RT-PCRSpecific ABCC11 amplified PCR products were detected in both experiment and control samples of human sweat glands.ABCC11 genotypingThe genotype of the ABCC11 SNP 538 was sequenced after RT-PCR of sweat gland samples from two groups.11 GA heterozygotes and 1 GG homozygote were identified in experiment group. All the control samples were AA homozygotes.The expression of the ABCC11 transcriptThe expression of the ABCC11 transcript was investigated by RT-PCR analysis of sweat gland samples from two groups. In contrast, expression of the housekeeping gene GAPDH and PolR2A gene were present in both sample types, suggesting that ABCC11 expression is specific in tissue containing apocrine sweat glands. In apocrine gland tissue, ABCC11 expression in experiment group was statistically significant notably above the expression in control group, controled with GAPDH/PolR2A (p-value=3.813e-07, wilcoxon test).ConclusionsIn our study, ABCC11 SNP 538G>A contributed to disfunction of axillary odor. That GA heterozygote and GG homozygote showed higher expression of ABCC11 gene of AA homozygote.Part Ⅱ The study on osmidrosis surgical treatment:Axillary Osmidrosis Treatment Using an Aggressive Suction-Curettage TechniqueIntroductionThe apocrine sweat gland plays the major role in the formation of human axillary odour. However, the physiological function of human apocrine sweat is poorly understood, although in numerous higher mammals, apocrine odour molecules serve as messengers in olfactory communication, for example, to attract mates, to signal danger (fight or flight) and to mark territory. Strong axillary malodour in humans is generally perceived as socially offensive in most cultures and is a troublesome problem for the individuals concerned. The clinical term for this condition is osmidrosis.Because profuse sweating and axillary malodor can lead to significant limitations in personal social interaction, especially in Asian countries, various methods of treatment have been introduced. In China, some patients prefer surgical therapy as a permanent solution as compared with nonsurgical methods such as laser treatments and botulinum toxin injections. In the past 10 years, open excision of the apocrine glands was considered the most effective and popular way of treating osmidrosis, however, it was often associated with complications (skin necrosis and hematoma) and noticeable postoperative scars.In recent years, axillary suction-curettage has become a popularminimally invasive procedure.Alarge series of studies from Korea has established it as a safe and effective surgical treatment for osmidrosis. However, the recurrence rate after treatment and effectiveness of suction-curettage remains contraversial. Since 2009, our center has introduced the axillary suction-curettage technique as a treatment option and has performed the procedure on 65 patients. Herein, we present our technique as well as results and complications.Materials and MethodsPatientsFrom February 2009 to February 2014, more than 300 patients who received bilateral axillary operation became candidates. Those who had already received surgical treatment (including laser) or had scars at the axillary area were excluded. Sixty five patients (130 axillae) who requested for invisible scars were enrolled into the experimental group. The control population was historical, non-concurrent in this study. Only candidates who could be paired with the experimental members were enrolled into the control group. Parings were done in two aspects:the same gender and age (±2 years). The study obtained approval from the Committee on Medical Ethics of the hospital. Informed consent was obtained from every patient.Surgical Procedure1) Experimental group procedure:Preoperative markings were made. After tumescent anesthesia, a special cannula, connected to the liposuction machine was inserted into the subcutaneous tissue and set to work. First, a routine suction (at 1.875 mmHg) was done to make multiple subcutaneous channels. The axillary skin was pinched by surgeon’s fingers to wrap the skin round the cannula and then aggressive scrapings and suctions were done. The cannula was rotated through 90 degrees to ensure curettage of the skin. Suction-curettage was done repeatedly until thinning of the skin was observed. The whole procedures took an average of 15 minutes. Irrigation was done to get rid of free tissue. The incision was left without sutures. The tissues aspirated were sent for pathological examination. Patients were asked to put on special clothing for 7days. Showering and excessive activity using the shoulders were also prohibited.2) Control group procedure:Local anesthesia was given. After skin and subcutaneous incision was made, two flaps whose pedicles were at the edge of dissection lacuna were created. The flaps were turned over with the fingertips and subcutaneous tissue was trimmed. The subdermal vascular network was conserved to reduce the risk of circulation compromise of the flap. After coagulation, the incision was closed with 6-0 monofilament sutures. A bolus dressing and an elastic bandage were used for 7days. Showering and excessive activity using the shoulders were prohibited for 1 week.Post-operation evaluationFollow-up was done on individual basis, via outpatient visits or telephone enquiries, on 1st 3rd and 6th months after operation. Five aspects were evaluated: patient satisfaction, skin necrosis, hematoma, malodour recurrence and scars.StatisticsData were analyzed with SPSS (19.0). Chi square test and Fisher’s exact test was used as statistical methods for data analysis, and p-value<.05 was defined as statistically significant.ResultsGender ratio (F:M=42:23) was equal in both experimental and control groups. Ages ranged from 15 to 38 years old (average age 22.78 years) and 16 to 39 years (average age 22.91 years) respectively. Follow-up in experimental group ranged from 3 to 40 months (average 7.69months), while 3 to 20 months (average 6.81months) in control group.Patient SatisfactionTwenty two patients (33.85%) in the experimental group and 8 patients (12.31%) in the control group were totally satisfied. Compared with the control group, patients in the experimental group were more satisfied with the outcome of the procedure (p-value=0.004, Chi square test).Skin NecrosisSkin necrosis in the axillae was observed in three patients (2.31%) in the experimental group as compared with 16 patients (12.31%) in the control group. Only one patient in the experimental group (left axillae) experienced severe axillary necrosis, and received local skin flap as treatment. Eight patients in the control group were diagnosed with severe axillae necrosis, and were re-operated on (re-suturings in 3 axillae, local skin flap in 5 axillae). Full recovery took about 14 days. The experimental group had statistically significant lower incidence of skin necrosis (p-value=0.002, Chi square test).HematomaAxillae hematoma was reported in 3 patients (2.31%) in control group had with none in the experimental group. However, the results were not statistically significant (p-value=0.25, Fisher’s exact test).RecurrenceSymptom recurrence was reported in 15 (11.54%) and 6 (4.62%) in the experimental and control groups respectively. Among these cases, only two patients with 3 axillae in the experimental group and one with 2 axillae in the control group requested for secondary treatment, while most patients confirmed the surgery had significantly alleviated the malodor. The results are statistically different, but not significant (p-value=0.041, Chi square test).ScarsThe control group had longer and more ugly scars (p-value=0.000, Chi square test), while the scars in experimental group were less visible.ConclusionsThe aggressive suction-curettage technique was used on 65 patients with axillary osmidrosis. Patients who underwent this procedure (aggressive suction-curettage) showed lower necrosis rate (1.88%, p<0.01), higher patient satisfaction (33.85%, p<0.01), higher recurrence rate (11.54%, p<0.05) and less ugly scars (0.77%, p<0.01), compared with those who underwent the excision procedure. To conclude, the aggressive suction-curettage procedure appears to be a reliable choice of treatment in patients diagnosed with axillary osmidrosis.