The Study On Molecular And Clinical Characteristics Of Hereditary Adrenal Diseases

Background:The adrenal gland is one of the most important endocrine organs in the human body with complex and important functions.The adrenal gland is composed of two distinct structures-the medulla and the cortex.The adrenal medulla is in the center of the adrenal gland,accounting for only 10% of the whole adrenal gland.It mainly secretes catecholamines such as epinephrine and norepinephrine.It is regulated by the sympathetic nerve and plays important roles in emergency situations.The adrenal cortex can be further divided into 3 layers,the zone reticular,zone fascicular,and zone glomerulosa(from inside to outside).The innermost zona reticularis mainly synthesizes adrenal androgens from cholesterol through a series of enzymatic reactions and then secrete them,which are regulated by both the hypothalamic-pituitary-adrenal(HPA)axis and the hypothalamic-pituitary-gonadal axis.The increase of adrenal androgens mainly leads to premature adrenarche and virilization.The zona fascicular accounts for about 75% of the adrenal cortex and mainly secretes mainly cortisol.Its functions include the regulation of glucose,protein and fat metabolism,and immune system.It is regulated by the HPA axis.Increased cortisol leading to moon face,central obesity,purple lines,and osteoporosis(Cushing syndrome);severe cortisol depletion can lead to life-threatening adrenal crisis.Finally,the zona glomerulosa,which accounts for 15% of the adrenal cortex,produces mineralocorticoid-mainly aldosterone,which regulates blood pressure and water and electrolyte balance.It is regulated by the renin-angiotensin-aldosterone system.Excess aldosterone can lead to hypertension,hypokalemia,and even irreversible cardiovascular damage.Due to the complexity of adrenal anatomy and function,adrenal diseases often manifest as different syndromes,so its diagnosis and differential diagnosis are difficult.With the application of next-generation sequencing,many adrenal diseases are considered to be closely related to genetics,which can be familial and appear in the form of syndrome.In adrenal medullary diseases,about 30%-40% of pheochromocytoma patients carry germline gene mutations,which can manifest as part of a variety of complex tumor syndromes according to the different mutant genes,such as VHL syndrome and MEN2 syndrome.The three layers of the adrenal cortex(zona reticularis,zona fasciculata,and zona glomerulosa)also have their corresponding genetic disorders,such as: congenital adrenal hyperplasia(CAH)caused by CYP21A2 and other genes,primary bilateral macronodular adrenal hyperplasia(PBMAH)caused by ARMC5 and other genes,primary pigmented nodular adrenocortical disease(PPNAD)caused by PRKAR1 A,and familial hyperaldosteronism caused by germline KCNJ5 mutations.Lack of the understanding of the molecular mechanism and clinical characteristics of these rare genetic diseases can easily lead to missed diagnosis,misdiagnosis,and insufficient treatment in the process of diagnosis and treatment.By studying the molecular and clinical characteristics of hereditary pheochromocytoma from the inner medulla to the outer cortex(CAH,PPNAD and PBMAH),this article provides important theoretical and practical significance for in-depth understanding of the pathophysiology and molecular mechanism of this kind of genetic diseases,and improves the level of diagnosis and clinical decision-making.Materials and methods :A total of 1383 cases of adrenal diseases(adrenal cortical adenoma,cortical carcinoma,cortical hyperplasia and(ectopic)pheochromocytoma)diagnosed by pathology or imaging from 2000 to 2022 were retrospectively collected.Among them,51 patients with early onset,bilateral/multiple lesions and family history of genetic diseases were screened.Peripheral blood samples of the patient were collected for sequencing to identify the susceptible gene mutations,and Sanger sequencing was used to verify the mutations in the proband and his family members.The clinical data,laboratory examination results,and imaging examination results of the patients and their family members were collected.Results and conclusions:1.Clinical studies of hereditary pheochromocytoma/paraganglioma and associated syndromesFifteen cases of PCC/ PGL-related syndromes were diagnosed by next-generation sequencing and clinical diagnosis,including 7 von Hippel-Lindau(VHL)syndromes,3MEN2 syndromes and 5 familial PGL syndromes.By analyzing the molecular and clinical features of VHL syndrome,we found that:(1)a total of 27 VHL syndrome patients and 1asymptomatic carrier were screened in 7 VHL syndrome families;239G>T and c.444＿457del are associated with VHL2 B,and c.293A>G is associated with VHL2 C.Two patients with recurrent PCC and multiple renal cell carcinoma received sunitinib targeted therapy,and the treatment effect was significant.(2)Seven patients and three asymptomatic carriers were identified from three families with multiple endocrine neoplasia(MEN)type 2syndrome.1901G>C,c.1832G>A and c.1901G>A mutations were all associated with MEN2 A.Prophylactic thyroidectomy is of great significance for these patients.(3)There were 4 patients with SDHB,1 patient with SDHD mutation,4 patients with truncated mutation(SDHB: c.343C>T,c.268C>T;SDHD: c.337＿340del),1 patient with pathogenic point mutation;SDHx mutations are closely related to PGL of ectopic pheochromocytoma,which is mostly located in the retroperitoneal paraganglion.In conclusion,mutations in different genes and site are closely associated with different syndrome manifestations.The application of NGS technology has deepened our understanding of the molecular mechanism of hereditary PCC/ PGL-related clinical syndromes,and has played an important role in clinical accurate diagnosis,follow-up and genetic counseling.2.Genotype-presentation correlations in patients with CAHIn this part,three families with CYP21A2 mutation and one family with rare CYP17A1 mutation were screened.By analyzing its molecular and clinical features,we found that:(1)The phenotypes caused by CYP21A2 and CYP17A1 mutations are similar but also significantly different.Proband 1 and 4 had CYP21A2(c.293-13C>G/c.518T>A,p.I173N)and CYP17A1(c.1118A>T,p.H373L/c.1459＿1467del9,p.D487＿F489del)mutations,respectively.Bilateral or unilateral adrenal myelolipoma could be present in both families.However,proband 4 and two sisters of the family who carried compound heterozygous mutations of CYP17A1 caused increased synthesis of corticosterone hormones and mainly presented with hypokalemia and other symptoms.The proband 1 and his brother presented with androgen excess symptoms such as short stature due to the mutation.The proband 2 was A girl carrying 30kb-loss/c.[188A>T;518T>A],p.[H63L;I173N] of CYP21A2,the latter one were novel double mutant,which belonged to simple virilizing associated mutations and presented with typical feminization.The diagnosis was made at birth due to abnormal vulvar examination and elevated 17-OHP.The proband 1(male)and his brother mainly presented with giant bilateral adrenal myelolipoma and short stature.The proband 3(male,c.293-13C>G/c.518T>A,p.I173N)presented with short stature and azoospermia caused by hypogonadotropic hypogonadism.(3)The same compound heterozygous mutation can also lead to different phenotypes.Probands 1 and 3 had the same two compound heterozygous mutations of CYP21A2,but their phenotypes were different.In conclusion,different genes and gender can lead to differences in phenotypes,and the same compound mutation can also produce different phenotypes.Genetic testing has a certain clinical significance in the etiological diagnosis of CAH(especially atypical CAH patients),timely and accurate treatment,disease prevention,and genetic counseling.3.Molecular and clinical features of rare hereditary ACTH-independent adrenal nodular hyperplasiaThree patients with hereditary ACTH-independent adrenal nodular hyperplasia were identified by clinical features and genetic testing.A comparison of its molecular and clinical features found that:(1)Both PBMAH and PPNAD can cause severe Cushing’s syndrome.Case 1(39-year-old male)and case 2(51-year-old female)had PBMAH,and case 3(16-year-old male)with PPNAD.Laboratory tests showed elevated cortisol,ACTH suppression,and negative high-dose/low-dose dexamethasone suppression tests.The clinical manifestations were severe Cushing’s syndrome.(2)Patients with PBMAH mainly presented with bilateral adrenal nodular hyperplasia,and the nodules were generally large(>10mm),but PPNAD often occurred at an earlier age,with small adrenal nodules and hyperpigmentation.Case 3 had skin and sclera hyperpigmentation.(3)ARMC5 mutations were an important molecular feature of PBMAH.Cases 1 and 2 carried nonsense mutations in ARMC5,one of which was a novel pathogenic mutation(p.Arg173*).PPNAD is closely related to Carney syndrome caused by PRKAR1 A mutation.Case 3 carried PRKAR1 A pathogenic mutation.PBMAH may be associated with or without somatic mutations of ARMC5.ARMC5 patients with somatic mutations have an earlier age of onset and more severe symptoms.In conclusion,detailed analysis of the molecular characteristics of ACTH-independent adrenal nodular hyperplasia is helpful to reduce missed diagnosis and misdiagnosis,and is of great importance for the diagnosis and treatment of patients.