Vol. 56 No. 1/2009, 83–88 Regular paper

Hyperparathyroidism (pHPT) is a relatively frequent endocrinopathy, however, the molecular mechanisms of its etiology remain poorly understood. This disorder is mainly associated with benign tumours (adenoma) and hyperplasia of the parathyroid, hence, the focus is directed also to genes that are likely to be involved in carcinogenesis. Among such genes are ErbB/Her family genes already used in diagnosis of other tumours (e.g., breast carcinoma) and reported also to play a role in development of endocrine lesions. So far, ErbB-1/Her-1/EGFR expression has been detected in pHPT-associated adenomas and hyperplasia as opposed to no expression in normal parathyroid tissue. Moreover, losses or gains of the fragments of chromosomes where ErbB/Her genes are located have been reported. In this study, the gene dosage of ErbB/Her family genes were determined for the first time in parathyroid adenomas, hyperplasia and morphologically unchanged tissue in order to establish their putative role in the development of the disease. Genomic DNA was isolated from 33 patients with sporadic hyperparathyroidism and the gene copy numbers were assessed using real-time PCR. The ErbB/Her genes' profile was unaltered in most of the examined samples. Two low-level amplifications of ErbB-1/Her-1/EGFR gene, two deletions of ErbB-2/Her-2, and six deletions of ErbB-4/Her-4 were found. The ErbB-3/Her-3 gene remained unaffected. No correlation with clinical parameters was found for any gene. Both the low number of alterations and a lack of their associations with clinical parameters exclude the prognostic value of the ErbB/Her genes family in parathyroid tumourigenesis. Nevertheless, the ErbB-4/Her-4 deletions seem to be interesting for further investigations, especially in the context of PTH secretion.

One of the genes which have already been examined in parathyroid benign tumours is that encoding ErbB-1/Her-1/EGFR.This protein is a member of the ErbB/Her family of tyrosine kinase receptors and among others is involved in cellular signal transduction (reviewed in Zaczek et al., 2005).Altered expression of ErbB/Her receptors might lead to impaired cell proliferation.ErbB/ Her genes are often mutated or have altered expression in cancer and precancerous cells, which is often a sign of progression and development of disease (Vogt et al., 1998;Xu et al., 2002;Awaya et al., 2005;Saxby et al., 2005;Sassen et al., 2008).ErbB/Her aberrations have also been reported in some types of endocrine lesions, e.g., of thyroid, pheochromocytoma and pancreas (Sworczak et al., 2002;Freudenberg et al., 2005;Saxby et al., 2005;Lee et al., 2007).ErbB-1/Her-1/EGFR expression has been found in pHPT-related adenomas and hyperplasia (Sadler et al., 1996;Gülkesen et al., 2001) and, interestingly, recent data have documented an involvement of its activation in the initiation of parathyroid hyperplasia as a consequence of secondary hyperparathyroidism (Cozzolino et al., 2005).
Hence, the aim of our study was to examine variations of the copy number of ErbB/Her genes in pHPT-related adenoma and hyperplasia as well as in morphologically unchanged parathyroid tissue.
Furthermore, the possible impact of chromosomal aberrations on the disease status was analyzed.

MATERIAl AND METHoDS
Surgical specimens.Human parathyroid tissue samples were collected from 33 patients from the Northern part of Poland, affected with sporadic pHPT.All patients had no history of familial hyperparathyroidism or a multiple endocrine neoplasia syndrome.The patients were diagnosed and treated in the Department of Endocrinology and Internal Medicine at the Medical University of Gdańsk (Poland) in years 2001-2006.After surgical removal tissue specimens were frozen and stored at -80°C.Material was classified by pathologist.A selected part of the material was used for DNA extraction.Ten out of 33 cases (30.3%) were classified as adenoma (AGP) and 23 (69.7%) as hyperplasia (HypGP).There were 24 females and 9 males among patients.The mean age at the time of surgery was 59.26 year (range: 29-77 year).Additional demographic and clinical information was available for 33 patients.The mean size of the maximal dimension of excised AGP and HypGP was 20.67 mm (range: 8-50 mm).All patients had increased levels of serum parathormon (PTH), calcium (Ca), creatinine and alkaline phosphatase and a decreased phosphate (P) serum concentration.Seven samples characterized as morphologically unchanged parathyroid obtained during thyroidectomy from patient not affected with hyperparathyroidism were taken as a control.
DNA isolation.DNA was isolated from 20 mg of selected parathyroid tissue with the usage of QIAamp® DNA Mini Kit (Qiagen, Germany) as described by the manufacturer.Control DNA was isolated in the same way from SKBR-3 (ATCC HTB-30) and MDA-MB-468 (ATCC HTB-132) cell lines.The purity of DNA was verified by the ratio A 260 /A 280 = 1.70-1.95.For further procedures DNA was prepared at concentration of 10 ng/µl and stored at -20°C.
Measurement of gene copy number by realtime PCR.A set of primers was designed for real-time PCR (sequences 5'→3') as follows: ErbB-1/Her-1/EGFR forward (F): TCTGCATTCCTGCCGAGTTC, ErbB-1/ Her-1/EGFR reverse (R): GCAGTCTCCACTC-CATGCTCA, ErbB-2/Her-2 F: CTGCTGGTCGTG-GTCTTGG, ErbB-2/Her-2 R: CTGCAGCAGTCTCCG-CATC, ErbB-3/Her-3 F: GCTCATCCTGGCCAACAC, ErbB-3/Her-3 R: CATACCCGATCAGCACCAGTG, ErbB-4/Her-4 F: TGCTGCT GCTCACTCATTGG, ErbB-4/Her-4 R: CTGCGCGTCTGGAGTTGTTG, 3p F: CTCATAGGCGAAGGCACCAG, 3p R: GGT-CAAGTTCCGCACACACC.Real-time PCR was performed on a LightCycler ver.2.0 (Roche, Germany).PCR reactions were prepared in a total volume of 10 µl using LC FastStart DNA Master SYBR Green I (Roche, Germany) as described by the manufacturer, with MgCl 2 at a final concentration of 2.5 µM and primers at a final concentration of 0.5 µM each.The DNA template was added at 10 ng per sample.The following temperature profile was used: denaturation -10 min at 95°C; quantification (40 cycles) -10 s at 95°C and 10 s at 60°C with single fluorescence acquisition; melting curve -0 s at 95°C, 15 s at 65°C and 0 s at 95°C with continuous fluorescence acquisition (ramping rate 0.1°C/s); cooling -30 s at 40°C.The ramping rate -if not mentioned otherwise -was set at 20°C/s.The Cp values and melting curves of all samples were obtained and analyzed.For every sample the mean of three Cp values for each gene was calculated into its concentration according to a standard curve prepared for each gene separately.Then, the ratio between the concentration of the gene of interest and a reference gene was counted and normalized to the related ratio of normal parathyroid tissue for the same gene.The gene dosage was compared to the cut-off values and defined as amplification (A), deletion (D) or normal gene copy number (N).
Statistics.Non-parametric Kruskal-Wallis test was performed to compare the results with the clinical data.The level of significance was set to 0.05 for a significant correlation.

RESulTS
In order to analyse the ErbB/Her family genes' dosages in pHPT the quantitative real-time PCR was established with the usage of region on chromosome 3p as a reference.A standard curve was obtained for each tested gene and the efficiency of reaction was checked.The Cp values of each gene from all samples were counted into dosage according to the appropriate standard curve.The cut-off values were determined as follows: below 0.5 as a deletion (D), 0.5-2.0 as a normal gene copy number (N) and above 2.0 as an amplification (A).According to these values, 10 parathyroid adenomas, 23 hyperplasia and 7 normal parathyroid tissues were examined for copy number of the ErbB/Her genes.
The control normal parathyroid tissue not affected with pHPT revealed normal copy numbers of all tested genes with one exception where, interestingly, an ErbB-4/Her-4 deletion appeared.The assessment of gene copy numbers for two cell lines with known amplified ErbB-1/Her-1/EGFR or ErbB-2/Her-2 gene (respectively, MDA468 and SKBR322 cell line) confirmed the method's accuracy and sensitivity.
The majority of pHPT-affected patients had a completely unchanged profile of the ErbB/Her genes.Six patients with pHPT (18.2%) revealed abnormalities of at least one examined gene.
Two low-level amplifications of ErbB-1/ Her-1/EGFR were found (6.1%), two (6.2%) deletions of ErbB-2/Her-2 and five (15.1%) deletions of ErbB-4/ Her-4.There were no amplifications of ErbB-2/Her-2 and no aberrations of ErbB-3/ Her-3.Simultaneous aberrations of two genes were found in three patients (9.1%).There were two cases with co-deletions of ErbB-2/ Her-2 and ErbB-4/Her-4 and one case with amplification of ErbB-1/Her-1/EGFR and deletion of ErbB-4/ Her-4.Alterations in more than two genes were found.The range and the mean of  the assessed gene dosages as well as the frequency of amplifications, deletions and normal gene copy numbers found for each gene in all examined samples are presented in Table 1.The clinical data of selected parathyroid samples with the Erb/Her alterations are presented in Table 2.No significant correlations were found between any gene's copy number and clinical parameters like patients' age, type of changes, size and localization, PTH, Ca, P and alkaline phosphatase level.However, an interesting tendency was observed in samples bearing an ErbB-4/Her-4 deletion.The concentration of serum PTH was decreased in those samples in comparison with samples with a normal ErbB-4/Her-4 gene copy number (P > 0.05) (Fig. 1).

DISCuSSIoN
pHPT is a frequently diagnosed endocrinopathy with severe consequences for the whole organism.The majority of diagnosed cases are associated with benign parathyroid tumours.Hence, genes known to be involved in tumourigenesis of other organs are tested as factors potentially related to the status of illness and possibly indicating the prognosis for patients.In this study, genes of the ErbB/Her family -known to play a role in tumourigenesis of other organs -were investigated for the first time in pHPT-related adenoma and hyperplasia as well as in morphologically unchanged parathyroid tissue.
The reference locus (on chromosome 3p) was chosen by analysis of the available CGH and FISH literature data on sporadic parathyroid adenomas and carcinomas.It is believed to be the least frequently altered region in the Caucasian race (Agarwal et al., 1998;Kytölä et al., 2000;Garcia et al., 2002).Those studies also show that regions where genes of the ErbB/Her family are located (chromosomes 2, 7, 12 and 17) reveal some gains (particularly chromosome 17) or losses.Nevertheless, these changes did not occur very often (Agarwal et al., 1998;Palanisamy et al., 1998;Garcia et al., 2002).In general, ErbB/ Her aberrations are detected mainly in patients af-fected with more advanced types of tumour.In the case of benign tumours, abnormalities are not observed (Brandt et al., 1995;Schwartz et al., 1998;Xu et al., 2002) or appear only very rarely (Vogt et al., 1998;Awaya et al., 2005;Dimova et al., 2006), but then they are correlated with the later stages and/ or progression of the disease (Awaya et al., 2005).
In our experiments most of the samples had unchanged gene profiles.The only detected amplifications were those of the ErbB-1/Her-1/EGFR gene.The hypothesis about ErbB-1/Her-1/EGFR involvement in parathyroid tumourigenesis has been suggested by other authors who performed in situ hybridization or immunohistochemistry (Sadler et al., 1996;Gülkesen et al., 2001), and recently also in secondary hyperparathyroidism as an initial event for hyperplasia (Cozzolino et al., 2005).Gülkesen et al. (2001) noticed also that ErbB-1/Her-1/EGFR overexpression might be related to young age of patients.In contrast, in our analysis both patients with the ErbB-1/Her-1/EGFR amplification were of the age of around 70.We did not find any relationship between these amplifications and other clinical parameters.The deletions of the ErbB-2/Her-2 gene found in two cases are not common in any type of tumours where mainly amplifications play an important role in carcinogenesis and are a prognostic factor (Sworczak et al., 2002;Xu et al., 2002;Awaya et al., 2005;Freundenberg et al., 2005).
The relatively high prevalence of deletions of the ErbB-4/Her-4 gene in our study is interesting.To the best of our knowledge, ErbB-4/Her-4 deletions have been detected very rarely in tumours (Zaczek et al., 2008).The reason of this apparent discrepancy might be the type of method used in previous studies.Real-time PCR used in our analysis is a very sensitive and precise tool enabling exact determination of gene dosage which was not possible with other methods.However, the losses on chromosome 2q (where ErbB-4/Her-4 is located) in parathyroid tissue reported in the literature might also support our results (Palanisamy et al., 1998;Kytölä et al., 2000).
The possible relationship between ErbB-4/Her-4 status and serum PTH should be investigated on ErbB/Her family genes copy number in hyperparathyroidism a larger group of patients.The present results can only signal a tendency which seems to be worthy of further interest in the context of hypertension related to pHPT and rising knowledge about the biological role of ErbB-4/Her-4.

CoNCluSIoNS
In the present study no relationship between the histological type of changes or other clinical data and Erb/Her genes' copy number alterations was found in patients affected with pHPT.Thus, no member of the ErbB/Her family can serve as prognostic factor in the studied disorder.Nevertheless, the presence of relatively numerous ErbB-4/Her-4 abnormalities among the examined patients suggests their possible role in pHPT.

Figure 1 .
Figure 1.Parathormon concentration in serum of patients affected with sporadic hyperparathyroidism and bearing deletion or normal gene copy number of ErbB-4/Her-4 gene.

Table 1 . The gene dosages of ErbB/Her family in 33 parathyroid tissues of the patients affected with sporadic primary hyperparathyroidism.
Note that 32 samples were tested for ErbB-2/Her-2 gene dosage.