VHL mutation as a cause of three generations familial pheochromocytoma

Background Pheochromocytoma is a rare disease, and its familial occurrence is quite uncommon. The aim of this paper is to report a three‑generation phenotypical expression of a case familial occurrence of pheochromocytoma. Case presentation A 25‑year‑old female, with a history of adrenalectomy for pheochromocytoma, arrived at the shock room during her third pregnancy with an adrenergic crisis and hypoglycemia. To prevent perinatal com‑ plications, the patient was stabilized and the newborn was delivered through a Kerr‑type cesarean section. A detailed history revealed that the paternal grandfather of the patient had an unilateral pheochromocytoma, whereas her paternal uncle had a bilateral pheochromocytoma. Additionally, a brother of the patient presented a unilateral pheochromocytoma. Amplicons for PCR assays were designed to span the protein‑coding segments of the three Von Hippel–Lindau ( VHL) exons, and the PCR products were sequenced using the Sanger method. In the trace of exon 3, we detected in the sample of the proband a heterozygous guanine to adenine transition (NM_000551.4 c. 552G > A) within the protein‑coding segment of exon 3 of the VHL gene, which leads to a substitution of the arginine residue at position 161 by a glutamine residue in the encoded peptide (NP_000542.1p.R161Q). This mutation was absent in two unaffected daughters. Conclusion A VHL mutation was suspected and confirmed in this family that was not transmitted to a fourth genera‑ tion. This case illustrates the importance of molecular genetics methodologies to assist genetic counseling in cases of pheochromocytoma where familial aggregation is presumed.


Background
Pheochromocytomas are rare tumors of chromaffin cells derived from the neural crest and mainly found in the adrenal medulla, although they can appear in other sites (paragangliomas).These are difficult to diagnose catecholamine-producing neuroendocrine tumors, and the familial aggregation of cases is quite uncommon.Most pheochromocytomas are isolated and apparently sporadic, while approximately one in ten cases occurs in the context of a syndrome (neurofibromatosis type I, Multiple Endocrine Neoplasia type II, Von Hippel-Lindau (VHL) syndrome), sometimes displaying Mendelian patterns of inheritance [1,2].
The annual incidence of pheochromocytomas is estimated around 2-8 cases per million [3,4], being responsible for less than 0.1-0.2% of hypertension cases without sex preference, and most are diagnosed between the fourth and fifth decade of life, although they can appear at any age [4,5].
About 80-85% of pheochromocytomas are found in the adrenal medulla, and the other 15-20% in extraadrenal areas, mainly the Zuckerkandl organ, although it can occur in other areas of the thorax and abdomen; extra-adrenal pheochromocytomas are more common in children [6,7].
Pheochromocytomas are classified as benign or malignant, the difference being that the malignant type is capable of metastasizing [1,3,5].The formation of pheochromocytomas has been linked to germline mutations in more than 20 loci, causing activation of three signaling pathways: (1) kinase signaling-related genes, (2) pseudohypoxic Krebs cycle-related genes and (3) Wnt signaling-related genes [1,6].
Diagnosis is based on confirmation of clinical suspicion by biochemical tests and image studies [2,8].Biochemical tests are indicated in asymptomatic patients with family history of pheochromocytoma or germline mutations associated with increased risk for pheochromocytoma as well as patients with either (1) signs or symptoms suggesting an excess of catecholamines, with or without arterial hypertension, (2) arterial hypertension that requires three or more antihypertensive drugs for its management, (3) unexplained blood pressure variability, (4) paradoxical response in surgical interventions after the use of anesthesia and beta adrenergic receptor blockers or (5) in the event of the incidental finding of an adrenal tumor [8,9].
The preferred diagnosis test is the determination of free metanephrines (normetanephrine and metanephrine) in urine and plasma [2,5].Plasma determination is considered superior to the urinary one, with a sensitivity of 97.9% and a specificity of 94.2% [8,10,11].The treatment of pheochromocytoma is based on a multidisciplinary intervention, including the management of symptoms such as hypertension, headache, kidney damage.However, the definitive treatment is surgery to remove the neuroendocrine tumor [5,8].
Pheochromocytoma is a rare disease, and its familial presentation is quite uncommon.The aim of this paper is to report a three-generation phenotypical expression of familial pheochromocytoma.

Case report
The proband, a 25-year-old female, was diagnosed with a pheochromocytoma located at the right adrenal gland and later submitted to right adrenalectomy, developing right renal atrophy after the procedure.She had a grandfather with unilateral pheochromocytoma, an uncle with bilateral pheochromocytoma and a brother with unilateral pheochromocytoma (Fig. 1), the last two treated surgically.
During her third pregnancy, the patient was received at the shock room with hypoglycemia, neuroglycopenic symptoms and fluctuating sleepiness.To avoid perinatal complications an emergency Kerr-type cesarean section was performed, obtaining a healthy female newborn.The patient had two previous pregnancies, one spontaneous and complete abortion at 3 weeks of gestation.During her second pregnancy, she had altered fasting glucose and severe urinary infections that led to sepsis, making necessary the obstetrical interruption with a satisfactory evolution for her and the product.Table 1 shows the main paraclinical studies.

Genetic analysis
Samples (1 mL of peripheral venous blood) were collected from the patient, her affected brother and her unaffected two daughters, in tubes containing ethylenediaminetetraacetic acid.Genomic DNA was isolated using the QIAamp DNA Mini Kit (Qiagen, Hilden, Germany), following directions from the manufacturer.Briefly, 400 µL of blood were mixed with 20 µL of proteinase K and 250 µL of lysis buffer and mixed.After an incubation at Fig. 1 Genealogy of the family affected by pheochromocytoma described in the present report.Individuals (I,1), (III,3) and the proband (III,2) had unilateral tumors, whereas individual (II,3) had a bilateral tumor 56ºC for 10 min, 300 µL of ethanol was added, transferring the mix to a DNA isolation column, which was spun to 6,000 g for 1 min twice, discarding the flow-through.The column was first washed with 500 µL of AW1 buffer and spun at 6,000 g for 1 min and washed again with 500 µL of buffer AW2, centrifuging it at 17,000 g for 3 min a first time and for 1 min a second time, discarding the flow-through.The DNA was eluted from the column to a 1.5-mL microfuge using 50 µL of elution buffer and spun at 6000 g for 1 min.Purity and concentration were determined by UV absorbance at 260 nm and 280 nm of wavelength and integrity by agarose gel electrophoresis.

Sanger sequencing
Amplicons for PCR assays were designed to span the protein-coding segments of the three VHL exons using primers at a final concentration of 3.2 µM.Exon 1 was amplified using primers VHL-1F (5'-acagtaacgagttggcctagc-3') and VHL-1R (5'-ttcagaccgtgctatcgtcc-3'), exon 2 was amplified using primers VHL-2F (5'-gtgtaggtcaggggaaatgg-3') and VHL-2R (5'-ggataacgtgcctgacatc-3'), and exon 3 was amplified using primers VHL-3F (5'-actacagaggcatgaacacc-3') and VHL-3R (5'-ACT TCT CTA ATG GGC AGG C-3' , capital letters denote exonic sequence).600 ng of genomic DNA was PCR-amplified using the GoTaq Green Master Mix (Promega, Madison, WI, USA), following directions from the manufacturer.An initial denaturation step was carried out at 96 °C for 3 min, followed by 30 cycles, each one with a denaturation step at 96 °C for 30 s, a annealing step at 60 °C for 30 s and an extension step at 72 °C for one minute.After the 30 cycles, a final extension step was performed at 72 °C for five minute.The PCR products were purified from excised 2.5% agarose gel bands using the QIAquick Gel Extraction Kit (Qiagen) and quantified with a Nanodrop spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA).50 ng of purified PCR product was used to generate 20 µL Sanger sequencing reactions with the BigDye Terminator kit v3.1 (Thermo Fisher Scientific).Sequencing reactions were in turn purified employing Centri-Sep columns (Thermo Fisher Scientific).Capillary electrophoresis was run in a Genetic Analyzer 310 (Thermo Fisher Scientific) following manufacturer's instructions.

Results
The PCR assays performed resulted in efficient amplification of the intended targets as revealed by the Sanger sequencing electropherograms.In all three samples screened (the proband, her affected brother and her two unaffected daughters), no variants were detected in the traces corresponding to exons 1 and 2 of the VHL gene.In the trace of exon 3, we detected in samples from the proband and her affected brother a heterozygous guanine to adenine transition (NM_000551.4 c.552G > A) within the protein-coding segment of exon 3 of the VHL gene, which leads to a substitution of the arginine residue at position 161 by a glutamine residue in the encoded peptide (NP_000542.1p.R161Q).Upon sequencing of the relevant VHL gene segment, we observed that this mutation was not present in two unaffected daughters of the proband (Fig. 2).All sequences were performed in both directions, being internally consistent.

Discussion
According to various studies, 10% of pheochromocytoma occurrences correspond to familial cases and reports of this tumor affecting several members of a family are uncommon.For example, a familial case consisting of a male patient and three affected daughters was registered among a series of 24 pheochromocytoma cases treated within a 15-year period in a referral center.Within this family, the most characteristic finding in these patients was the malignant hypertension.After the two daughters underwent subtotal adrenalectomy, they remained normotensive and with normal cortisol values [12].
The familial aggregation of pheochromocytoma cases has been described in other articles, including one of them reporting a case of a mother and daughter affected by this tumor and neurofibromatosis 1.Both underwent successful surgical resection and the daughter had healthy offspring [13].
Another group reported two further familial cases.Regarding the first of them, the proband had an extraadrenal tumor causing severe renal artery stenosis, with pheochromocytomas present in three successive generations of the family.The second familial occurrence included multiple pheochromocytomas cases associated with von Hippel-Lindau syndrome and a family member with multiple endocrine neoplasia type 2. These genetic entities and syndromes share peculiar interrelationships, pathologically related to an aberration in the migration, growth and differentiation of the neural crest cells, with a common neuroectodermal origin [14].
The tumor-suppressor protein pVHL, the product of the VHL gene, is a E3 ubiquitin ligase that forms a complex with Elongin B and C to mediate the degradation of HIF1α y HIF2α and among other functions; it also stabilizes the tumor-suppressor p53, enhancing its transcriptional activity [15,16].The mutation identified in the family studied here, Arg161Gln, is located within the p53 and Elongin C binding site (residues 157-172).Previously, it has been shown that the Arg161Gln mutation impairs the induction of apoptosis caused by p53 activation in comparison with the wild-type pVHL [16].

Conclusion
The majority of pheochromocytomas are related to abnormal gene expression of neuroectoderm-derived tissues, usually because of germline or somatic mutations affecting specific genes.This kind of tumor can occur as an isolated entity of as part of a monogenic syndrome.Isolated tumors can also display Mendelian patterns of inheritance when familial aggregation of cases occurs.Given the severity and often life-threatening nature of complications of pheochromocytomas, effective and accurate genetic counseling to affected families should be provided.Here, we describe an assay that aided us to provide genetic counseling in a three-generation pheochromocytoma family.
Abbreviation VHL Von Hippel-Lindau Fig. 2 Sanger sequencing traces obtained from samples from affected (III,2 and III,3) and unaffected (IV,2 and IV,3) subjects from the genealogy depicted in Fig. 1