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Pheochromocytoma

Germline Pathogenic Variants Identified by Targeted Next-Generation Sequencing of Susceptibility Genes in Pheochromocytoma and Paraganglioma

Sinem Yalcintepe1*, Hakan Gurkan1, Fatma Nur Korkmaz2, Selma Demir1, Engin Atli1, Damla Eker1, Hazal Sezginer Guler1, Drenusha Zhuri1, Emine Ikbal Atli1, Semra Ayturk Salt3, Mustafa Sahin2, Sibel Guldiken3

1Department of Medical Genetics, Faculty of Medicine, Trakya University, Edirne, Turkey;

2Department of Endocrinology and Metabolism, Faculty of Medicine, Ankara University, Ankara, Turkey;

3Division of Endocrinology, Department of Internal Medicine, Faculty of Medicine, Trakya University, Edirne, Turkey

Abstract

The aim of this study was to evaluate germline variant frequencies of pheochromocytoma and paraganglioma targeted susceptibility genes with next-generation sequencing method. Germline DNA from 75 cases were evaluated with targeted next-generation sequencing on an Illumina NextSeq550 instrument. KIF1B, RET, SDHB, SDHD, TMEM127, and VHL genes were included in the study, and Sanger sequencing was used for verifying the variants. The pathogenic/likely pathogenic variants were in the VHL, RET, SDHB, and SDHD genes, and the diagnosis rate was 24% in this study. Three different novel pathogenic variants were determined in five cases. This is the first study from Turkey, evaluating germline susceptibility genes of pheochromocytoma and paraganglioma with a detection rate of 24% and three novel variants. All patients with pheochromocytoma and paraganglioma need clinical genetic testing with expanded targeted gene panels for higher diagnosis rates.

Key words: paraganglioma, pheochromocytoma, targeted sequencing, susceptibility genes

Received: February 1, 2021; Accepted after Revision: February 28, 2021; Published: March 13, 2021.

Author for correspondence: Sinem Yalcintepe, MD, Department of Medical Genetics, Faculty of Medicine, Trakya University, Edirne, Turkey. Tel.: 00905377168691. Email: [email protected]

How to cite: Yalcintepe S et al. Germline Pathogenic Variants Identified by Targeted Next-Generation Sequencing of Susceptibility Genes in Pheochromocytoma and Paraganglioma. J Kidney Cancer VHL. 2021; 8(1): 19–24

Doi: http://dx.doi.org/10.15586/jkcvhl.v8i1.171

Copyright: Yalcintepe S et al.

License: This open access article is licensed under Creative Commons Attribution 4.0 International (CC BY 4.0). http://creativecommons.org/licenses/by/4.0

Introduction

Paraganglioma and pheochromocytoma are defined as neurogenic tumors arising from paraganglial cells found in neuroendocrine tissues (1). Paraganglionic tissues, on the other hand, are specialized cell groups and are generally found scattered throughout the body. These cells are mostly located in the adrenal medulla, aorta, vascular wall, heart, prostate, and ovaries (2). They are groups of cells in close proximity to the sympathetic and parasympathetic nervous system. Tumors originating from chromaffin cell groups are called pheochromocytomas. (3). Paragangliomas do not generally contain epinephrine and are not stained with chromium salts. Therefore, these tumors are considered chromaffin negative. These paraganglial cell groups are found in the carotid, jugular glomus, aorticopulmonary glomus, vagus, and ciliary glomus where chemoreceptors are found in the body (1).

Von Hippel–Lindau (VHL) Syndrome is a hereditary cancer syndrome involving more than one system, characterized by benign or malignant tumors and cystic lesions (4). Hemangioblastomas, renal cysts and kidney cancer, pheochromocytoma, pancreatic cysts and neuroendocrine tumors, endolymphatic sac tumors, and epididymal and ligamentum latum cysts may be seen in the brain, spinal cord, and retina (5). Retinal hemangioblastomas are usually the first sign of the disease and cause vision loss. Up to 70% of the affected individuals can be diagnosed with retinal hemangioblastomas detected around the age of 25 (6, 7).

Pheochromocytoma associated with a pathogenic variant occurs at a younger age than pheochromocytoma cases without any mutation (8). Although there is no clear age limit, having been diagnosed younger than 30–45 may indicate the presence of a mutation. SDHA, SDHAF2, SDHB, SDHC, SDHD, MAX, and TMEM127 genes are offered to be analyzed for the diagnosis of paraganglioma/pheochromocytoma (PGL/PCC) syndromes (9). For differential diagnosis, NF1, VHL, RET, and EPAS1 genes should be analyzed (10). In this study, a custom panel of

KIF1B, RET, SDHB, SDHD, TMEM127, and VHL genes were analyzed with a targeted gene panel using next-generation sequencing method for rapid and cost-effective diagnosis and differential diagnosis in patients with PGL/PCC. Only these six genes could be analyzed, as this study was planned retrospectively and this custom panel was used between the years 2013 and 2020 in our department; this custom panel was designed firstly in 2013 with only six genes. To our knowledge, this is the first study presenting germline pathogenic/likely pathogenic variants of paraganglioma and pheochromocytoma patients from Turkey.

Material and Methods

Patients

Seventy-five cases with a pre-diagnosis of PGL/PCC were examined in the Endocrinology Clinic and referred to the Medical Genetics clinic for molecular testing from August 2013 through November 2020. Cases were included for having high levels of blood or urinary catecholamines, metanephrines, methoxytyramine, or chromogranin A, and/or a suspicion of PGL/PCC on imaging tests, and/or personal or family history of PGL/PCC in our study. Pediatric cases and/or adult cases who have not had any imaging tests due to pregnancy, breastfeeding, claustrophobia, medical conditions, serious illness, and acute or chronic renal insufficiency were excluded from this study.

The cases had no relation except cases 4 and 5 who are brothers (Table 1).

Table 1: Demographic features, phenotypes, and genotypes of the cases with pathogenic/likely pathogenic variants detected in our study.

Case Age/Gender Phenotype Affected family member Gene Nucleotide change Protein dbSNP or HGMD ID
1 FA 40/F Pheochromocytoma Unknown VHL NM_000551.4:c.583C>T p.(Gln195Ter) rs5030825
2 MA 35/M Pheochromocytoma (Bilateral) Father, sister VHL NM_000551.4:c.481C>T p.(Arg161Ter) rs5030818
3 MAU 27/M Pheochromocytoma (Bilateral surrenal—metastatic lymph nodes) Unknown VHL NM_000551.4:c.277G>A p.(Gly93Ser) rs5030808
4 AGY 29/M Pheochromocytoma (Bilateral renal and eyes, pancreatic cysts)) Mother, brother (Case 5) VHL NM_000551.4:c.202T>C p.(Ser68Pro) CM073416
5 AY 32/M Pheochromocytoma (Right kidney-clear cell carcinoma) Mother, brother (Case 4) VHL NM_000551.4:c.202T>C p.(Ser68Pro) CM073416
6 MD 33/M Paraganglioma, liver cysts Cousin VHL NM_000551.4:c.202T>C p.(Ser68Pro) CM073416
7 MG 42/M MEN2A (Pheochromocytoma, medullary thyroid cancer, hyperparathyroid) No history RET NM_020975.6:c.1832G>A p.(Cys611Tyr) rs377767397
8 AB 62/M Medullary thyroid cancer No history RET NM_020975.6:c.1946C>T p.(Ser649Leu) rs148935214
9 UB 47/F Right thyroid lobe nodule, high serum calcitonin level No history RET NM_020975.6:c.2370G>T p.(Leu790Phe) rs75030001
10 FB 28/F Medullary thyroid cancer Unknown RET NM_020975.6:c.1901G>A p.(Cys634Tyr) rs75996173
11 HG 59/F Pheochromocytoma Unknown RET NM_020975.6:c.1900T>C p.(Cys634Arg) rs75076352
12 MK 22/M Paraganglioma Unknown SDHB NM_003000.3:c.508dupT p.(Tyr170LeufsTer9) novel
13 BB 15/M Paraganglioma No history SDHB NM_003000.3:c.298T>C p.(Ser100Pro) CM056397
14 HT 24/M Tumor located posterior to the liver segment No history SDHB NM_003000.3:c.262A>C p.(Thr88Pro) novel
15 AC 18/M Prevertebral paraganglioma, hypertrophic cardiomyopathy Unknown SDHB NM_003000.3:c.262A>C p.(Thr88Pro) novel
16 YB 30/M Pheochromocytoma (Right surrenal) No history SDHD NM_003002.4:c.147dupA p.(His50ThrfsTer19) rs876659130
17 BS 82/F Paraganglioma, medullary thyroid cancer No history SDHD NM_003002.4:c.326A>G p.(Gln109Arg) novel
18 MY 24/F Pheochromocytoma (Right surrenal) Grandmother-Hypertension SDHD NM_003002.4:c.326A>G p.(Gln109Arg) novel

dbSNP: database of single nucleotide polymorphisms, HGMD: The Human Gene Mutation Database.

The written informed consent forms were obtained from the patients or from their legal guardians. This study is approved by the Ethical Committee of our university with the number 2021/46 and performed in consonance with the principles of the Declaration of Helsinki.

Molecular analysis

Genomic DNA was isolated from peripheral blood samples with ethylenediaminetetraacetic acid tetrasodium salt dihydrate (EDTA) of the patients by using EZ1 DNA Investigator Kit (Qiagen, Hilden, Germany). Primary quality control of the isolated DNA samples was performed using NanoDrop (Thermo Fisher Scientific, Waltham, MA), and samples having A260/280 values between 1.8 and 2.0 were included in the study.

QIAseq Targeted DNA Panel (Qiagen, Hilden, Germany) was performed to analyze six genes (KIF1B, RET, SDHB, SDHD, TMEM127, and VHL). Libraries were prepared according to the manufacturer’s instructions. Quality control of the prepared libraries was applied with Qubit dsDNA BR Assay system (Invitrogen, Carlsbad, CA). Fastq files were performed on Illumina NextSeq550 (Illumina Inc., San Diego, CA, ABD). Libraries covering the target genes were prepared according to the QIAseq Targeted DNA Panel protocol (Qiagen, Hilden, Germany). Following the target enrichment process, libraries were sequenced on the Illumina NextSeq550 (Illumina Inc., San Diego, CA, ABD). QCI analysis (Qiagen, Hilden, Germany) was used for Quality control and ordering Variant Call Format file. Variant analysis was performed with Ingenuity software (Qiagen, Hilden, Germany).

For verifying the variants and segregation analysis, primers were designed for all needed regions, and Sanger sequencing was performed using an ABI 3130 (Applied Biosystems, USA) capillary electrophoresis system.

Variant classification

ACMG-2015 (American College of Medical Genetics) (11) guidelines were followed for the classification of all the variants, and recommendations of the Human Genome Variation Society (12) were followed to describe the novel variants. ClinVar (13), HGMD-Professionel 2020 database, and literature information were considered for collecting the information about known variants.

Results

Six different germline genes (KIF1B, RET, SDHB, SDHD, TMEM127, and VHL) were analyzed in 75 cases with a pre-diagnosis of PGL/PCC. These genes were covered by 241 amplicons with Custom QIAseq Paraganglioma Panel (CDHS-17364Z-241). Two hundred and thirty eight of 241 amplicons (99%) yielded sequence reads, with a mean depth of 950 × per amplicon and sample.

The cases had a mean age of 40.4 (38 female cases with a mean age of 46.5; 37 male cases with a mean age of 34.1). Five of the patients (27.7%) had an affected family member.

Eighteen cases (24%) had pathogenic/likely pathogenic variants in our study. Six cases had pathogenic/likely pathogenic variants in VHL gene-four different variants. Five cases had five different pathogenic/likely pathogenic variants in RET gene. Four cases had three different pathogenic/likely pathogenic variants (two novel variants) in SDHB gene. Three cases had two different pathogenic/likely pathogenic variants and one novel variant in SDHD gene. We detected three different novel pathogenic/likely pathogenic variants in this study.

Discussion

In the current study, we designed a targeted NGS assay for susceptibility genes to analyze the pheochromocytoma and/or paraganglioma. SDHB, SDHD, RET, and VHL genes showed pathogenic variants in our study. Five of 18 cases (27.7%) were diagnosed with a novel pathogenic/likely pathogenic variant. Prevalence of germline mutations is an important consideration for offering molecular diagnostics.

In this study, six cases was diagnosed with VHL pathogenic variants and the younger patient with VHL pathogenic/likely pathogenic variant was 27 years old (Table 1, Case 3). He was diagnosed with metastatic bilateral surrenal adenomas. A study analyzing 86 unselected PGL/PCC tumor samples reported that two pathogenic variants were detected in VHL gene (14). VHL gene was also reported in 19.1% of the cases as the reason for bilateral pheochromocytoma (15). Fifty percent of our VHL cases had bilateral pheochromocytoma, one case had also eye involvement and pancreatic cysts, and one case had liver cysts (Table 1).

In the current study, five cases had different pathogenic RET variants with different clinical presentations as pheochromocytoma, medullary thyroid cancer, and thyroid nodules. The ages of these cases were between 28 and 62 years. Medullary thyroid cancer is a rare tumor originating from the parafollicular C cells of the thyroid, and it is characterized by RET proto-oncogene mutation in almost all hereditary cases and more than 40% of sporadic cases (16). In addition to clinical findings in medullary thyroid cancer evaluation, RET proto-oncogene mutation screening is highly recommended to determine genotype-phenotype risks and to distinguish between sporadic and familial cases in terms of maintenance of the disease. RET polymorphic alleles were reported as an additive effect on the estimated risk of age-related pheochromocytoma penetrance in MEN2 patients (17). Pheochromocytoma in MEN2 patients is usually reported as bilateral and rarely to be metastatic (18). It was reported that analysis of patients with RET 634 mutations with and without pheochromocytoma showed that pheochromocytoma was not associated with a more advanced stage of medullary thyroid cancer at diagnosis or a shorter survival (18). We detected codon 634 mutations in two cases (40%) of five RET-mutated cases—C634Y-mutated case had medullary thyroid cancer, and C634R-mutated case had pheochromocytoma. Similarly with the literature, our cases had mostly codon 634 mutations. As a limitation of this single-center study, the current cohort was limited. Codon 634 mutations were reported as the most frequent RET mutations in Brazil (19). C634Y (63.4%), C634R (28.2%), C634W (3.8%), C634G (3.1%), C634S (1.1%), and C634F (0.4%) mutations were reported in the same study (19). Codon 634 mutations were considered to be associated with an aggressive clinical course (20).

A 24-year-old female in the current study was diagnosed with right surrenal adenoma with a novel pathogenic variant NM_003002.4:c.326A>G on SDHD gene (Table 1, Case 18). The same variant was detected in another 82-year-old female who has no relation with the abovementioned 24-year-old female (Table 1, Case 17). Another SDHD pathogenic variant was detected in a 30-year-old male who had right surrenal adenoma (Table 1 case 16). SDHD and SDHB genes were reported as the most important causative genes of hereditary PGL/PCC in Asia when patients are tested with multi-gene NGS panel (21). In a case from Turkey, an 81-year-old female was reported with multinodular goiter and essential hypertension had NM_003002.3:c.325C>T (Gln109Term) pathogenic variant in SDHD gene (22).

Two different novel pathogenic SDHB variants were detected in four cases in our study. All these four cases involved young patients who were not related with each other. Distinct differences were reported in the clinical and histopathological characteristics between genetic variants in SDHB (23). Our two nonrelated cases had the same novel pathogenic variant (NM_003000.3:c.262A>C) in SDHB gene with different clinical presentations (Table 1, cases 14 and 15) (Figure 1). 58 tumor samples (55 PGL, including 45 head and neck PGL, 2 PCC, 1 GIST) were analysed in a study, and pathogenic variants in 50 patients (22 (13%) SDHB, 1 (3.2%) SDHC and 27 (57%) SDHD) were detected (24). We analyzed germline variants in this study, and the frequencies of the pathogenic variants were 5.3% and 4% for SDHB and SDHD genes, respectively.

Figure 1: IGV (Integrative Genomics Viewer) image of case 14 showing NM_003000.3:c.262A>C novel variant in SDHB gene.

In our study, SDHB carriers had no metastasis; only one case had hypertrophic cardiomyopathy, additively (Table 1, Case 15). Metastatic disease occurs in 2–23% of all paraganglioma cases and is a major cause of mortality (25). SDHB carriers were reported to have the highest risk for metastasis (26). Genetic counseling was given to these SDHB cases and their relatives for future risks, and they have been considered for clinical follow-up.

For SDHD carriers, only pathogenic variants inherited from the father will cause disease, due to maternal imprinting with silencing of the maternal allele occurs for SDHD (27). Three SDHD carriers were identified in our study, paternal analysis could not be planned due to not being alive.

KIF1B and TMEM127 were also included in our targeted gene panel, but any pathogenic/likely pathogenic variant was not detected in these genes. KIF1B is a rare reason for pheochromocytoma and TMEM127 is a susceptibility gene for PGL/PCC. We analyzed the germline variants in the current study. Probably, analyzing somatic variants would support the detection of KIF1B and TMEM127 variants, too.

Conclusion

To our knowledge, this is the first study from Turkey, analyzing germline KIF1B, RET, SDHB, SDHD, TMEM127, and VHL variants together. A study from Turkey reported 18 RET C634G mutations in 88 individuals (28), while another study reported VHL mutations in two pheochromocytoma patients (29). On the other hand, our study has limitations, especially the targeted genes in our study. We could not include other paraganglioma genes like SDHA, SDHC, MAX, etc., and we did not include NF1 carrier cases in this study. Further studies with larger cohorts and larger gene panels are needed for determining the prevalence and genotype-phenotype correlations for PGL/PCC patients.

Conflict of Interest

The authors declare that there is no conflict of interest.

Acknowledgements

The authors are grateful to the participants.

REFERENCES

1. Guilmette J, Sadow PM. A guide to pheochromocytomas and paragangliomas. Surg Pathol Clin. 2019 Dec;12(4):951–65. 10.1016/j.path.2019.08.009

2. Kiernan CM, Solórzano CC. Pheochromocytoma and paraganglioma: Diagnosis, genetics, and treatment. Surg Oncol Clin N Am. 2016 Jan;25(1):119–38. 10.1016/j.soc.2015.08.006

3. Farrugia FA, Martikos G, Tzanetis P, Charalampopoulos A, Misiakos E, Zavras N, et al. Pheochromocytoma, diagnosis and treatment: Review of the literature. Endocr Regul. 2017 Jul 1;51(3):168–81. 10.1515/enr-2017-0018

4. Sanford T, Gomella PT, Siddiqui R, Su D, An JY, Bratslavsky G, et al. Long term outcomes for patients with von Hippel-Lindau and pheochromocytoma: Defining the role of active surveillance. Urol Oncol. 2021;39(2):134.e1–e8. 10.1016/j.urolonc.2020.11.019

5. Fields FR, Suresh N, Hiller M, Freed SD, Haldar K, Lee SW. Algorithmic assessment of missense mutation severity in the Von-Hippel Lindau protein. PLoS One. 2020 Nov 5;15(11):e0234100. 10.1371/journal.pone.0234100

6. Kreusel KM. Ophthalmological manifestations in VHL and NF 1: Pathological and diagnostic implications. Fam Cancer. 2005;4:43–47. 10.1007/s10689-004-1327-0

7. Dollfus H, Massin P, Taupin P, Nemeth C, Amara S, Giraud S, et al. Retinal hemangioblastoma in von Hippel-Lindau disease: A clinical and molecular study. Invest Ophthalmol Vis Sci. 2002;43:3067–74.

8. Ma X, Li M, Tong A, Wang F, Cui Y, Zhang X, et al. Genetic and clinical profiles of pheochromocytoma and paraganglioma: A single center study. Front Endocrinol (Lausanne). 2020 Dec 11;11:574662. 10.3389/fendo.2020.574662

9. Irwin T, Konnick EQ, Tretiakova MS. Malignant intrarenal/renal pelvis paraganglioma with co-occurring SDHB and ATRX mutations. Endocr Pathol. 2019 Dec;30(4):270–5. 10.1007/s12022-019-09594-1

10. Else T, Greenberg S, Fishbein L. Hereditary paraganglioma-pheochromocytoma syndromes In: Adam MP, Ardinger HH, Pagon RA, et al. editors. GeneReviews®. Seattle, WA: University of Washington; 2008 [Updated 2018 Oct 4], p. 1993–2020.

11. Richards S, Aziz N, Bale S, Bick D, Das S, Gastier-Foster J, et al. Standards and guidelines for the interpretation of sequence variants: A joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology. Genet Med. 2015 May;17(5):405–24. 10.1038/gim.2015.30

12. den Dunnen JT, Dalgleish R, Maglott DR, Hart RK, Greenblatt MS, McGowan-Jordan J, et al. HGVS recommendations for the description of sequence variants: 2016 update. Hum Mutat. 2016 Jun;37(6):564–9. 10.1002/humu.22981

13. Landrum MJ, Lee JM, Benson M, Brown GR, Chao C, Chitipiralla S, et al. ClinVar: Improving access to variant interpretations and supporting evidence. Nucleic Acids Res. 2018 Jan 4;46(D1):D1062–7. 10.1093/nar/gkx1153

14. Welander J, Andreasson A, Juhlin CC, Wiseman RW, Bäckdahl M, Höög A, et al. Rare germline mutations identified by targeted next-generation sequencing of susceptibility genes in pheochromocytoma and paraganglioma. J Clin Endocrinol Metab. 2014 Jul;99(7):E1352–60. 10.1210/jc.2013-4375

15. Kittah NE, Gruber LM, Bancos I, Hamidi O, Tamhane S, Iñiguez-Ariza N, et al. Bilateral pheochromocytoma: Clinical characteristics, treatment and longitudinal follow-up. Clin Endocrinol (Oxf). 2020 Sep;93(3):288–95. 10.1111/cen.14222

16. Fagin JA, Wells SA, Jr. Biologic and clinical perspectives on thyroid cancer. N Engl J Med. 2016;15(375):1054–67. 10.1056/NEJMra1501993

17. Siqueira DR, Ceolin L, Ferreira CV, Romitti M, Maia SC, Maciel LM, et al. Role of RET genetic variants in MEN2-associated pheochromocytoma. Eur J Endocrinol. 2014 Jun;170(6):821–8. 10.1530/EJE-14-0084

18. Thosani S, Ayala-Ramirez M, Palmer L, Hu MI, Rich T, Gagel RF, et al. The characterization of pheochromocytoma and its impact on overall survival in multiple endocrine neoplasia type 2. J Clin Endocrinol Metab. 2013 Nov;98(11):E1813–19. 10.1210/jc.2013-1653

19. Maciel RMB, Camacho CP, Assumpção LVM, Bufalo NE, Carvalho AL, de Carvalho GA, et al. Genotype and phenotype landscape of MEN2 in 554 medullary thyroid cancer patients: The BrasMEN study. Endocr Connect. 2019 Mar 1;8(3):289–98. 10.1530/EC-18-0506

20. Febrero B, Rodríguez JM, Ríos A, Segura P, Pérez-Sánchez B, Torregrosa N, et al. Prophylactic thyroidectomy in multiple endocrine neoplasia 2 (MEN2) patients with the C634Y mutation: A long-term follow-up in a large single-center cohort. Eur J Surg Oncol. 2019 Apr;45(4):625–30. 10.1016/j.ejso.2018.09.002

21. Ting KR, Ong PY, Wei SOG, Parameswaran R, Khoo CM, Deepak DS, et al. Characteristics and genetic testing outcomes of patients with clinically suspected paraganglioma/pheochromocytoma (PGL/PCC) syndrome in Singapore. Hered Cancer Clin Pract. 2020 Dec 11;18(1):24. 10.1186/s13053-020-00156-9

22. Korkmaz FN, Gokcay Canpolat A, Bilezikci B, Gurkan H, Erdogan MF. A patıent wıth an atypıc neck mass lesıon. Acta Endocrinol (Buchar). 2020 Apr–Jun;16(2):232–5. 10.4183/aeb.2020.232

23. Main AM, Rossing M, Borgwardt L, Grønkær Toft B, Rasmussen ÅK, Rasmussen UF. Genotype-phenotype associations in PPGLs in 59 patients with variants in SDHX genes. Endocr Connect. 2020 Aug;9(8):793–803. 10.1530/EC-20-0279

24. Saie C, Buffet A, Abeillon J, Drui D, Leboulleux S, Bertherat J, et al. Screening of a large cohort of asymptomatic SDHx mutation carriers in routine practice. J Clin Endocrinol Metab. 2020 Nov 28:dgaa888. 10.1210/clinem/dgaa888

25. Mannelli M, Castellano M, Schiavi F, Filetti S, Giacchè M, Mori L, et al. Clinically guided genetic screening in a large cohort of Italian patients with pheochromocytomas and/or functional or nonfunctional paragangliomas. J Clin Endocrinol Metab. 2009 May;94(5):1541–7. 10.1210/jc.2008-2419

26. Turkova H, Prodanov T, Maly M, Martucci V, Adams K, Widimsky J Jr, et al. Characterıstıcs and outcomes of metastatıc SDHB and sporadıc pheochromocytoma/paraganglıoma: An natıonal ınstıtutes of health study. Endocr Pract. 2016 Mar;22(3):302–14. 10.4158/EP15725.OR

27. Muth A, Crona J, Gimm O, Elmgren A, Filipsson K, Stenmark Askmalm M, et al. Genetic testing and surveillance guidelines in hereditary pheochromocytoma and paraganglioma. J Intern Med. 2019 Feb;285(2):187–204. 10.1111/joim.12869

28. Algun E, Abaci N, Kosem M, Kotan C, Koseoglu B, Boztepe H, et al. Clinical characteristics and genetic screening of an extended family with MEN2A. J Endocrinol Invest. 2002 Jul–Aug;25(7):603–8. 10.1007/BF03345083

29. Dagdeviren Cakir A, Turan H, Aykut A, Durmaz A, Ercan O, Evliyaoglu O. Two childhood pheochromocytoma cases due to von Hippel-Lindau disease, one associated with pancreatic neuroendocrine tumor: A very rare manifestation. J Clin Res Pediatr Endocrinol. 2018 Jun 1;10(2):179–82. 10.4274/jcrpe.5078