Somatic Mutations In Chinese Han Aldosterone-producing Adenoma Patients

Objective: Recent studies indicate that somatic and germline mutations play a criticalrole in the development of aldosterone-producing adenoma. These mutations involvesuch genes as KCNJ5, ATP1A1, ATP2B3and CACNA1D. However, there is still nostudy of large sample size in China. The present study is to investigate the frequencyand expression of these genes in Chinese han APA patients, and to analyze theassociation of mutation status with clinical data and to provide new basis for clinicaldiagnosis and therapy of APA. Materials and Methods:1.Total tissue DNA wasexstrated from114APA patients and tissue RNA and peripheral blood DNA wereextracted from87APA patients. All the RNA were reversely transcripted into cDNA.All the DNA and cDNA were amplificated by PCR and sequenced for target genes todetect possible mutations and then to analyze the association of mutation status withclinical data.2. RT-PCR and immunohistochemistry were applied to investigate theexpression of KCNJ5mRNA and protein in APA tissues.3. The function of the twonovel KCNJ5mutations were verified in HEK293and H295R cells. Cell proliferationand membrane voltage were detected by MTS methods and DiSBAC2dye on HEK293cells. Aldosterone production and related genes expression were detected byradioimmunoassay kit and RT-PCR, respectively. Results:1.86out of114patientscarry the mutation of KCNJ5(75.44%), including reported mutations like G15R, L168Rand T158A, and two novel mutations. One patients presented with point mutationE147Q and insertion mutation simultaneously, whereas another had a36-baseduplication, G153_G164dup. No mutation was detected in the hotspots of ATP1A1andATP2B3and only one male patient was detected with a novel CACNA1D mutation,V748I.2. Compared with other studies, male and female patients had similar KCNJ5mutation rates (75.47%vs.74.19%), and mutation carriers had higher preoperativesystolic blood pressure and plasma aldosterone concentration, but lower preoperative potassium level and postural aldosterone responses than the non-mutation group.3. ThemRNA expression of KCNJ5and CYP11B2was higher in the KCNJ5mutated APAtissues. Immunohistochemistry results show that KCNJ5mutated tissues have greaterdisparity of staining than non-mutation tissues. Tissues without KCNJ5mutation alsohave a positive but uniform staining of KCNJ5protein. While non-functional adrenaltumors present negative staining of KCNJ5protein.4. Functional test showed that twonovel KCNJ5mutations could result in decreased proliferation and membrane voltagedepolarization in HEK293cells. More DiSBAC2accumulated in HEK293cellsexpressing mutated KCNJ5protein, which suggest that these cells had more depolarizedmembrane voltage. The transfection of KCNJ5-M1(with point mutation and insertionmutation simutanously) and KCNJ5-M2(with duplication mutation) increased thesecretion of aldosterone in H295R cells by2.27times and2.10time respectively. Afterstimulated by angiotension II for24h, the production of aldosterone increased by1.40times and1.34times. The KCNJ5-M1transfected cells showed a3.00-fold and3.09-fold increase in the mRNA expression of CYP11B1and CYP11B2, whereasKCNJ5-M2transfected cells showed a2.90-fold and2.94-fold increase in the mRNAexpression of the two genes. Conclusions:1. Chinese han APA patient have a highermutation frequency of KCNJ5gene than others countries, while no mutations weredetected in ATP1A1and ATP2B3genes.2Chinese male APA patients have a similarKCNJ5mutation rate with female patients.3.Mutated APA tissues have a higher mRNAexpression of KCNJ5and CYP11B2gene. KCNJ5protein expression tend to be moreheterogenous in KCNJ5mutated tissues while homogenous in wild-type KCNJ5tissues,while non-mutated tumor tissues have a negative staining of KCNJ5protein.4. The twonovel KCNJ5mutations could result in decreased cell proliferation, depolarizedmembrane voltage and increased aldosterone secretion and expression of related genes.