Introduction

There exist common genetic etiologies of tumor development among the endocrine glands, which is exemplified by inherited tumor-susceptibility syndromes such as multiple endocrine neoplasia (MEN) type 1 and 2, causing tumors of e.g., the parathyroid, and adrenal medulla (pheochromocytoma) [1]. Parathyroid tumors causing primary hyperparathyroidism (pHPT) is one of the more common endocrine tumors with a 2.3% prevalence in postmenopausal females [2]. Studies have so far identified genetic alterations in the cyclin D1 oncogene, the tumor suppressor genes MEN1 (located at chromosome 11q13), and HRPT2 (located at chromosome 1q25-q32), as pathogenically important for development of sporadic parathyroid tumors [3–6]. LOH at chromosome 11q13 is the most common chromosomal aberration in parathyroid tumors occurring in about 40% of sporadic tumors. However, only about 15–19% of cases display somatic mutations in the MEN1 gene [7–9]. This has lead to the speculation that another, yet identified, tumor suppressor gene on chromosome 11q13 is involved in parathyroid tumor development [10].

Recently, a novel putative neuroendocrine tumor suppressor gene located in the vicinity of the MEN1 gene was isolated. The SDHAF2 gene (also known as SDH5 or PGL2) encodes succinate dehydrogenase complex assembly factor 2 (SDHAF2), a highly conserved cofactor of flavin adenine dinucleotide (FAD) [11]. A missense mutation of the SDHAF2 gene was identified in a Dutch kindred with neuroendocrine paraganglioma of the head and neck. Functional analyses of the inactivating mutation (232G → A; Gly78Arg) showed complete loss of both flavination of SDHA and activity of the succinate dehydrogenase complex [11]. This SDHAF2 gene mutation also displayed parent-of-origin expression phenotype, with onset of tumor development only via the paternal line. SDHAF2 seems to have a role in flavination of SDHA, and correct flavination of SDHA is essential for a functional succinate dehydrogenase complex. Germline mutations in genes encoding succinate dehydrogenase subunits (SDHD, SDHC, or SDHB; formerly known as PGL1, PGL3, and PGL4) cause familial paraganglioma of the head and neck, as well as the thorax and abdomen (termed pheochromocytoma when occurring in the adrenal medulla) [12–15]. The enzyme complex plays a central role in cellular energy metabolism as both a mitochondrial tricarboxylic acid (TCA) cycle enzyme and as the complex II component of the electron transport chain. The role of TCA enzymes as tumor suppressors has broadened since the identification of the SDHD gene in 2000 [14], with mutations of fumarate hydratase reported as the cause of hereditary renal-cell cancer and leiomyomatosis [16], and the identification of somatic genetic aberrations of isocitrate dehydrogenase genes in glioblastoma [17].

In recent years, some groups have focused their attention toward mitochondrial DNA (mtDNA) and its role in the predisposition of cancer, especially among slowly progressing neoplasms. Costa-Guda et al. [18] analyzed and proved that within a majority of sporadic parathyroid adenomas there was indeed mutations present in the mtDNA. The mutations of this DNA have been implicated in the malfunction of the respiratory chain within the mitochondrial complex, particularly complex I. The chain can and is directly affected by reactive oxygen species (ROS), which have been implicated in the damage of DNA especially in slower growing tumors, such as breast, parathyroid, and papillary thyroid carcinoma [19]. The SDHAF2 gene through its role of flavination of SDHA is intricately involved within the electron transport chain of mitochondria [12–15].

In order to examine the role of SDHAF2 gene also in parathyroid tumorigenesis, we demonstrate that the gene is expressed in parathyroid tissue. Mutational analysis, however, failed to identify somatic genetic alteration of the gene, suggesting that mutations of the SDHAF2 tumor suppressor gene are unlikely to frequently contribute to parathyroid tumor development in sporadic pHPT.

Results

SDHAF2 gene expression in human parathyroid tissues

In order to determine whether the SDHAF2 gene is expressed in parathyroid tissue, total RNA was isolated from snap-frozen normal parathyroid cells and adrenal medulla (control). Reverse-transcriptase PCR of the cDNA revealed the expected size fragment in cDNA from normal parathyroid tissue and adrenal medulla (Fig. 1a), and verified by automated sequencing. Additionally, we demonstrated SDHAF2 gene expression in another cohort of 14 sporadic parathyroid adenomas from patients with PHPT and three normal parathyroid tissues (Fig. 1b).

Fig. 1
figure 1

a The novel endocrine tumor suppressor gene, SDHAF2, is expressed in parathyroid cells. Total RNA (10 μg) was isolated from snap-frozen parathyroid tissue and adrenal medulla (control). The expected product is visualized on a 1% agarose gel. Lanes 1 and 8, marker; lane 2, reverse transcriptase; lane 3, cDNA from normal parathyroid cells; lane 4, negative control (H20); lane 5, reverse transcriptase; lane 6, cDNA from adrenal medulla; and lane 7, negative control (H20). b. The novel endocrine tumor suppressor gene, SDHAF2, is expressed in parathyroid cells. Total RNA (10 μg) was isolated from snap-frozen parathyroid tissue both diseased and normal. The expected product is visualized on a 1% agarose gel. Lanes 1 through 14 represent parathyroid Adenomas, and lanes 15–17 are normal parathyroid tissue. Lanes marked “L” denote the DNA Ladder and the “N” is the negative control lane

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Direct sequence analysis of SDHAF2 in parathyroid tumors

Eighty patients with biochemically proven pHPT due to a single parathyroid adenoma were included in the study to examine whether tumor-specific somatic mutations were present in the SDHAF2 gene. The clinical characteristics of the patients are presented in Table 1. Automated sequencing of all coding regions identified an annotated SNP, rs879647 (A to G substitution in intron 2) in 9/80 parathyroid tumors, as well as in the germline DNA of these patients (Fig. 2). The frequency with which the various alleles were observed was as follows, the G allele was observed in 0.127 of the patients which is not significantly different from the 0.161 allele frequency reported in the HapMap CEU European cohort of 112 individuals (http://www.ncbi.nlm.nih.gov/projects/SNP/snp_ref.cgi?rs=879647). Similarly, the A allele had a similar observed frequency of 0.873 compared to 0.839 in the HapMap CEU European cohort (non significant). No tumor-specific somatic mutational aberrations, such as nonsense, frameshift, or other inactivating mutations were identified.

Table 1 Clinical characteristics of the 80 patients with primary hyperparathyroidism
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Fig. 2
figure 2

Identification of an annotated SNP (A to G substitution in intron 2) of the SDHAF2 gene in parathyroid adenomas of patients with primary hyperparathyroidism

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Discussion

Allelic loss at chromosome 11q13 is one of the most common genetic alterations in parathyroid tumors of pHPT, occurring in approximately 40% of tumors. However, only about 15–19% of parathyroid tumors exhibit somatic mutations in the MEN1 gene of likely importance to parathyroid tumorigenesis [7–9]. A yet to be identified tumor suppressor gene located on chromosome 11q13 also involved in parathyroid tumor development has thus been postulated [10]. The SDHAF2 gene is a novel putative endocrine tumor suppressor gene in the vicinity of the MEN1 gene (Fig. 3). Based on its function in regulating succinate dehydrogenase and the presence of a missense germline mutation in a family with cervical paragangliomas, SDHAF2 may represent an endocrine tumor suppressor [11]. We demonstrate within our study that the SDHAF2 gene is expressed in parathyroid cells; however, the role of its presence within parathyroid tissue has yet to be defined. We believe that the SDHAF2 gene may be an important candidate tumor suppressor gene that could acquire loss of function mutations specifically in the parathyroid tumor genome, leading to a selective advantage and parathyroid tumor development.

Fig. 3
figure 3

Location of the SDHAF2 gene on chromosome 11q, a region of frequent allelic loss in parathyroid adenoma, and it relation the MEN1 gene

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Our approach was designed to rigorously test the possibility that SDHAF2 serves as a classic tumor suppressor in the parathyroid glands. Only recurrent inactivating mutations can provide this critical evidence for a driving role of tumorigenesis [20]. We explored the mutational status of the SDHAF2 gene and failed to identify somatic mutations in any of the 80 parathyroid tumors studied. Although our study did not examine the possibility of epigenetic changes or mutations in non-protein coding regions of the gene, it would be unprecedented if inactivating alterations in an authentic tumor suppressor gene were to be found exclusively in these regions without ever appearing in the coding region.

In conclusion, our observations suggest that SDHAF2 acts infrequently, if at all, as a tumor suppressor gene in the molecular pathogenesis of parathyroid adenomas of pHPT.

Materials and methods

Subjects and tumor samples

Biopsies of normal human parathyroid glands were obtained from normocalcemic patients operated for atoxic goiter. These were biopsied due to macroscopic ambiguity of the microscopic diagnosis. Eighty patients with operatively verified, non-familial pHPT due to a single adenoma were included into the study. All the tumors were carefully evaluated by an experienced endocrine pathologist prior to use in the study. Tumors were dissected prior to processing to minimize contamination by normal cells. The tissue was snap frozen in liquid nitrogen and stored at −80°C. None of the patients demonstrated signs of familial hypercalcemia, clinical, and biochemical signs of multiple endocrine neoplasia syndromes nor had a history of previous neck irradiation. Blood was collected after an overnight fast and serum (s-) calcium (reference range 2.20–2.60 mmol/l) and intact s-PTH (reference range 10–65 ng/l) were determined preoperatively. All the patients had postoperatively reversed hypercalcemia during follow-up for at least 6 months. Informed consent was obtained and the study was approved by the institutional review board.

DNA, Total RNA, cDNA synthesis

High-molecular-weight genomic DNA from whole blood, and tumor tissue was isolated by standard methods as previously described [21]. Total RNA from snap-frozen tumor and normal tissue was isolated using the Nucleobond AX RNA Isolation Protocol (Machney-Nagel, Bethlehem, PA) [22]. RNA was not treated with DNAase during the extraction process. cDNA synthesis was performed using the iScript cDNA synthesis kit (Biorad, Hercules, CA).

RT-PCR

PCR reactions were performed using the mRNA specific primers 5′-CAGTGTTCTCGACTTCGTCGC-3′ and 5′-AGCGTCTGAATGATGTCACAC-3′. The PCR conditions were: one cycle of activation at 95°C followed by 30 cycles of amplifications at 95°C for 20 s, 60°C for 20 s, and 72°C for 20 s and a final elongation step at 72°C for 7 min using 50 ng of parathyroid cDNA and 15 pmol of each primer. The PCR products were analyzed on a 1% agarose gel to control the quality of products and exclude unspecific PCR reactions. PCR products were then purified using QIAquick PCR purification kit (Qiagen, Valencia CA) and sequenced by direct sequencing methods.

DNA sequencing of the SDHAF2 gene

The coding regions of the SDHAF2 gene were amplified using the primers in Table 2. The PCR conditions were: one cycle of activation at 95°C followed by 35 cycles of amplifications at 95°C for 20 s, 60°C for 20 s, and 72°C for 20 s and a final elongation step at 72°C for 7 min using 2.5 ng genomic DNA and 15 pmol of each primer. The PCR products were visualized on agarose gel to control the quality of products and exclude unspecific PCR reactions. The PCR products were then purified using QIAquick PCR purification kit (Qiagen, Valencia CA). Each sample (40 ng of DNA) was prepared for submission in 96-well Eppendorf twin tec plates (Eppendorf, Westbury, NY) in accordance with the high volume plate submission policies and procedures at the Yale Cancer Center/W.M. Keck Foundation Biotechnology Resource Laboratory. The sequencing reactions utilized fluorescently-labeled dideoxynucleotides (Big Dye Terminators) and Taq FS DNA polymerase in a thermal cycling protocol. The sequence analysis is carried out on 3,730 capillary instruments (Applied Biosystems Foster City, CA).

Table 2 Forward and reverse primers for amplification of all exons of the SDH5 gene
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Data analysis

Data are analyzed with the use of Sequencing Analysis and AutoAssembler software (Applied Biosystems Foster City, CA), and publically available web based resources (NCBI, GenBank). Statistical analysis was done using SPSS 16 (SPSS Inc, Chicago, IL).