Genetic investigation by next generation sequencing of primary aldosteronism caused by unilateral or bilateral adrenal hyperplasia

Author(s)

Marcela Rassi da Cruz

• Document type: Doctoral Thesis
• Press: São Paulo.
• Institution: Universidade de São Paulo (USP). Faculdade de Medicina (FM/SBD)
• Defense date: 2020-09-16

Examining board members

Madson Queiroz de Almeida; Luciano Ferreira Drager; Cláudio Elias Kater; Leonardo Vieira Neto

• Advisor: Madson Queiroz de Almeida

Abstract

Introduction: Primary aldosteronism (PA) is the most common form of endocrine arterial hypertension with an estimated prevalence of approximately 4% in hypertensives treated in primary care services and 10% in patients referenced to tertiary hospitals. The most frequent causes of PA are bilateral adrenal hyperplasia (BH) and aldosteronomas. Unilateral adrenal hyperplasia (UH) is a rare cause of PA, as well as familial PA. In the past few years, considerable advances have been made towards the pathogenesis of primary aldosteronism (PA). Somatic pathogenic variants in KCNJ5, CACNA1D, ATP1A1, ATP2B3 and CTNNB1 genes were identified in aldosteronomas. Among familial PA, germlines defects in KCNJ5, CACNA1D, CACNA1H and CLCN2 were identified in PA patients with early onset of PA. Despite the advances made towards the better understanding of PA pathogenesis, the genetic factors involved in PA caused by UH and BH in adults remain poorly elucidated. Aim: To search for new susceptibility genes in patients with PA caused by UH or BH. The specific aims of this study were: 1) To investigate somatic variants in KCNJ5, ATP1A1, ATP2B3 and CTNNB1 genes in PA caused by UH or BH; 2) To perform exome sequencing (paired blood and adrenal tissue) of PA patients caused by UH or BH; 3) Validate and perform functional studies of genetic variants in new candidate genes. Methods: We included 11 patients with PA caused by adrenal hyperplasia (5 UH and 6 BH). The presence of chimeric CYP11B1/CYP11B2 gene was ruled out in all cases. Then, we performed whole exome sequencing (HiSeq 2500, Illumina) in paired blood and adrenal tissue from five patients with UH and six with BH. All hyperplastic tissues were positive for CYP11B2 staining. We searched for rare germline coding variants (MAF < 0.01% in gnomAD) in ion-channel genes expressed in normal adrenal or in those previously associated with adrenal hyperplasia (such as phosphodiesterases, PDEs). Results: Somatic pathogenic variants in KCNJ5 previously described (p.Gly151Arg e p.Leu168Arg) were identified in 2 BHs. Interestingly, we also identified 4 germline heterozygous variants predicted to be pathogenic in silico in PDEs genes: 1) The p.Ala59Val in PDE1A in a patient with UH; 2) The p.Ile629Val in PDE2A in a patient with BH; and 3) The p.Arg217Gln and p.Gly392Val variants in PDE3B in two patients with BH. Next, PKA activity was analyzed in frozen tissue. PKA activity was increased in adrenal hyperplastic tissue from patients with germline variants p.Ile629Val PDE2A and p.Gly392Val PDE3B compared to normal adrenal. PDE2A staining was strong in zona glomerulosa and in nodules of hyperplastic areas. In vitro studies showed that PDE2A expression was decreased in HEK 293T cells transfected with p.I629V PDE2A vector compared to those transfected with WT PDE2A vector. Similarly, PDE3B expression was decreased in cells transfected with p.Arg217Gln or p.Gly392Val PDE3B vector. Additionally, PDE2A and PDE3B variants increased gene and/or protein expression of SGK1 and SCNN1G/ENaCg, downstream mediators of aldosterone signaling. Regarding patients with UH, we identified a novel germline heterozygous variant (c.5428-3C > T) in CACNA1H gene in one patient with UH. In addition, a rare missense heterozygous variant predicted to be pathogenic in silico (p.Leu2095Ile) in CACNA1I gene, which encodes the alpha 1I subunit of Cav 3.3 channel, was identified in a patient with UH. Conclusions: This evidence suggest that PDE2A and PDE3B variants might be involved with the pathogenesis of BH and expand the spectrum of genetic etiologies for PA. In addition, a rare germline and pathogenic in silico variant in CACNA1I was identified in a patient with PA caused by UH (AU)

• FAPESP's process: 17/13394-8 - Genetic investigation by next generation sequencing of Primary Aldosteronism caused by Unilateral or Bilateral Adrenal Hyperplasia
• Grantee: Marcela Rassi da Cruz
• Support Opportunities: Scholarships in Brazil - Doctorate (Direct)