Molecular and clinical genetic studies of a novel variant of familial hypercalcemia

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(10) Dissertation for the degree of Doctor of Philosophy (Faculty of Medicine) in Surgery presented at Uppsala University in 20002 ABSTRACT Szabo, E. 2002. Molecular and clinical genetic studies of a novel variant of familial hypercalcemia. Acta Universitatis Upsaliensis. Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine 1147. 56pp. Uppsala. ISBN 91-554-5300-7 Familial primary hyperparathyroidism (HPT) is a rare disorder that is treated surgically and mostly occurs in association with tumorsusceptibility syndromes, like multiple endocrine neoplasia and the hyperparathyroidism-jaw tumor syndrome. Familial hypercalciuric hypercalcemia (FHH) is another cause of hereditary hypercalcemia that generally is considered to require no treatment and is genetically and pathophysiologically distinct from HPT. Inactivating mutations in the calcium receptor gene cause FHH, whereas the down-regulated expression of the CaR in HPT never has been coupled to CaR gene mutations. Family screening revealed a hitherto unknown familial condition with characteristics of both FHH and HPT. The hypercalcemia was mapped to a point mutation in the intracellular domain of the CaR gene that was coupled to relative calcium resistance of the PTH release by transient expression in HEK 294 cells. Unusually radical excision of parathyroid glands was required to normalise the hypercalcemia. The mildly enlarged parathyroid glands displayed hyperplasia with nodular components. Frequent allelic loss on especially 12q was found and contrasts to findings in HPT. Allelic loss was also seen in loci typical for primary HPT like 1p, 6q and 15q, but not 11q13. Quantitative mRNA analysis showed that the glands had mild increase in a proliferation index (PCNA/GAPDH mRNA ratio) and mild reduction in genes important to parathyroid cell function, like CaR, PTH, VDR and LRP2. A previously unrecognized variant of hypercalcemia is explored that could be one explanation for persistent hypercalcemia after apparently typical routine operations for HPT. It also raises the issue of possibilities to treat FHH with parathyroidectomy provided it is radical enough. Keywords: hyperparathyroidism, calcium receptor, hypercalcemia, allelic loss, autosomal inheritance Eva Szabo, Department of Surgical Sciences, University Hospital, SE-751 85 Uppsala, Sweden © Eva Szabo 2002 ISSN 0282-7476 ISBN 91-554-5300-7.

(11) ARTICLES This thesis is based on the papers listed below, which are referred to in the text by their Roman numerals. I.. Carling T, Szabo E, Bai M, Ridefelt P, Westin G, Gustavsson P, Trivedi S, Hellman P, Brown E M, Dahl N, Rastad J. Familial hypercalcemia and hypercalciuria caused by a novel mutation in the cytoplasmic tail of the calcium receptor. J Clin Endocrinol Metab 85(5): 2042-47, 2000. II.. Szabo E, Hellman P, Lundgren E, Carling T, Rastad J. Parathyroidectomy in familial hypercalcemia with clinical characteristics of primary hyperparathyroidism and familial hypocalciuric hypercalcemia. Surgery 131(3): 257-63, 2002. III.. Szabo E, Carling T, Hessman O, Rastad J. Loss of heterozygosity in parathyroid glands of familial hypercalcemia with hypercalciuria and point mutation in calcium receptor. Submitted for publication. IV.. Szabo E, Westin G, Åkerström G, Rastad J, Carling T. Parathyroid messenger RNA levels in familial hypercalcemia with hypercalciuria and calcium receptor mutation. Submitted for publication. Reprints were made with permission from the publishers.. 4.

(12) CONTENT ABBREVIATIONS THE PARATHYROID GLANDS Physiology SPORADIC PRIMARY HYPERPARATHYROIDISM Pathophysiology SECONDARY HYPERPARATHYROIDISM FAMILIAL HYPERPARATHYROIDISM Multiple endocrine neoplasia The hyperparathyroidism - jaw tumor syndrome Familial isolated hyperparathyroidism FAMILIAL HYPOCALCIURIC HYPERCALCEMIA Calcium receptor mutations PARATHYROID TUMOR GENETCS IN SPORADIC HYPERPARATHYROIDISM AIMS OF THE STUDY MATERIAL & METHODS Persons & tumors Biochemistry in blood and urine Parathyroid histology Immunostaining DNA & RNA isolation and cDNA synthesis Sequence analysis Site-directed mutagenesis & transient receptor expression Measurement of intracellular calcium Loss of heterozygosity Real-time quantitative PCR Linkage analysis Statistical analysis. 5. 7 8 8 9 10 12 12 12 13 13 14 16 18 20 21 21 21 22 22 23 23 24 24 25 26 26 27.

(13) RESULTS & DISCUSSION Mutational characteristics Clinical characteristics Parathyroidectomy procedure Loss of heterozygosity Quantification of relative mRNA levels GENERAL SUMMARY ACKNOWLEDGEMENTS REFERENCES. 6. 28 28 29 32 34 36 38 39 41.

(14) ABBREVIATIONS 1,25(OH)2D3 ANOVA Ca2+ Ca/CrCl CaR cDNA DNA FHH FIHPT GAPDH HEK 293 HPT HPT-JT LDL LOH LRP2 MEN mRNA PCNA PCR PRAD 1 PTH Rb SEM VDR. Active vitamin D Analysis of variance Calcium ions Calcium to creatinine clearance Calcium receptor Complementary deoxy ribonucleic acid Deoxy ribonucleic acid Familial hypocalciuric hypercalcemia Familial isolated hyperparathyroidism Glyceraldehyde 3-phosphate dehydrogenase Human embryonic kidney cells Hyperparathyroidism The hyperparathyroidism - jaw tumor syndrome Low density lipoprotein Loss of heterozygosity Low density lipoprotein receptor-related protein2 Multiple endocrine neoplasia Messenger ribonucleic acid Proliferating cell nuclear antigen Polymerase chain reaction Parathyroid adenomatosis gene 1 Parathyroid hormone Retinoblastoma Standard error of the mean Vitamin D Receptor. 7.

(15) THE PARATHYROID GLANDS A majority of individuals have four parathyroid glands, two superior and two inferior that are symmetrically located in the neck in about 80% of persons. About 13% have one or more supernumerary glands 1. The superior parathyroid glands are mostly located above the intersection of the recurrent nerve and the inferior thyroid artery. The inferior ones are more variable in location and often situated on the lower thyroid pole 2. The average weight of a normal parathyroid gland is less than 60 mg in 95% of cases 3. Histologically, the parenchyma consists of chief and oxyphil parathyroid cells that are interspersed with variably abundant fat cells 2. PHYSIOLOGY Calcium ions (Ca2+) have profound biological effects and work both as cofactor for intracellular enzymes, like phospholipases and proteases, and as second messenger in several cellular functions 4-6. Extracellular Ca2+ concentrations are precisely regulated by active vitamin D (1,25(OH)2D3) and parathyroid hormone (PTH). PTH consists of 84 amino acids and is secreted by the parathyroid cells in response to lowered extracellular Ca2+. Recently, the thymus was described as an auxiliary source of PTH in parathyroid deficient mice 7. However, the significance of this PTH production and its physiological role are not known. PTH increases Ca2+ levels by stimulating osteoclastic bone resorption and Ca2+ reabsorption in the kidney tubules 8. Additionally, PTH stimulates 1-alfa-hydroxylase activity in the proximal kidney tubules, which increases synthesis of active vitamin D and thereby the intestinal Ca2+ absorption 9. Active vitamin D mainly acts via the vitamin D receptor (VDR). It seems to potentiate the effects of PTH on bone and kidney and to exert powerful negative feedback on PTH secretion and parathyroid cell proliferation 10-13. There is an inverse sigmoidal relationship between extracellular Ca2+ and PTH, and the steepest part of the dose-response curve is around the physiological Ca2+ concentration 8, 14, 15. Indeed the hormone secretion of the 8.

(16) parathyroid cells responds immediately to minimal changes in the Ca2+ concentration. Besides stimulating the secretion rate, hypocalcemia also increases PTH mRNA levels. The resulting increase in the amount of hormone secreted per cell seems to be accompanied by recruitment of quiescent cells that become secretory active 16. The calcium receptor (CaR) is an extracellular Ca2+-sensing receptor on the parathyroid gland surface that binds external Ca2+ and activates phospholipase C and production of inositol phosphate 17, 18. CaR belongs to the G protein-coupled receptor family and is expressed also in eg. brain, kidney tubules and thyroid C-cells 19, 20. Another protein LRP2 (megalin) has also been implicated in calcium sensing and regulation of PTH secretion, since an antibody recognizing this protein can inhibit extracellular Ca2+ mediated inhibition of the PTH secretion 21-24. LRP2 belongs to the LDL receptor family and may be essential for uptake of 25 hydroxyvitamin D3 24, 25.. SPORADIC PRIMARY HYPERPARATHYROIDISM. Primary hyperparathyroidism (HPT) affects about 1% of the adult population. It is characterised by hypercalcemia and high levels of PTH usually due to tumor development in the parathyroid glands 26-28. Biochemically, patients with primary HPT usually display, apart from hypercalcemia, hypercalciuria, relative phosphatemia and hypomagnesemia. The prevalence rises with age in both sexes and up to 3% of elderly females display biochemical signs consistent with HPT 26-28. In addition, an autopsy examination of individuals without substantial renal diseases revealed histological abnormalities of the parathyroid glands in 10% of both sexes 29. Clinical presentation of HPT has changed over time. Historically, it was accompanied by pronounced renal, skeletal and mental symptoms, while it nowadays is characterised by more subtle manifestations 30. Such manifestations include fatigue, anxiety, irritability and apathy that can be interpreted as being appropriate for age in elderly. 9.

(17) individuals 31, 32. Moreover, HPT may be associated with overrepresentation of cardiovascular risk factors like hypertension, diabetes mellitus and dyslipidemia 32-34. Indeed, there have been suggestions from Scandinavia that premature death in cardiovascular diseases can occur even in mild HPT 35. About 85% of patients with sporadic primary HPT has a single enlarged parathyroid gland (adenoma), while mainly chief cell hyperplasia of two or more glands is found in the others 2, 36. Parathyroid carcinoma is seen in 1% to 3% of the patients with primary HPT 37. Sporadic HPT is treated with extirpation of the enlarged gland/glands. Formal subtotal parathyroid resection leaving a remnant equivalent to the size of a normal gland is very seldom required even in multiglandular disease. More than 95% of the patients become normocalcemic after parathyroidectomy with use of the strategy outlined above, which sometimes is referred to as a "conservative operative approach". PATHOPHYSIOLOGY Most, if not all, parathyroid adenomas are monoclonal tumors 3840 . Parathyroid hyperplasia can be diffuse, nodular or a variable mixture of the two. Diffuse and nodular parathyroid hyperplasia is seen in sporadic primary and secondary HPT and in some tumor susceptibility syndromes and consists to some extent of monoclonal lesions, which may arise from polyclonal hyperplasia 41-45 . Enlarged parathyroid glands leading to clinically detectable HPT invariably contain an increased mass of parenchymal cells. Active vitamin D, via its receptor VDR, can decrease both PTH mRNA production and parathyroid cell proliferation, whereas low Ca2+ and low active vitamin D stimulates parathyroid cell proliferation 46-48,49. Investigations of the parathyroid cell proliferation with immunohistochemical staining of the cell cycle markers Ki67 and p27Kip1 show the highest proliferation rate in parathyroid carcinomas, intermediate values in hyperplasia of secondary HPT and similarly low values in adenoma and hyperplasia of primary HPT 49, 50. Additionally, an increased. 10.

(18) proportion of hormonally active cells has been suggested to contribute to the relative PTH excess, since both PTH mRNA and intracellular protein levels per cell unit have been found to be decreased in primary and secondary HPT 51. The combined excess of PTH and Ca2+ depends on relative insensitivity to Ca2+ with a right shifted and flattened dose response curve 52. The increase in ED50 in the PTH - Ca2+ relationship is usually denoted as an increased set-point, ie. the extracellular Ca2+ level at which half-maximal inhibition of the PTH secretion is attained. Reduced expression of CaR mRNA and protein levels have been found in the majority of tumors in primary or secondary HPT and may contribute to the lowered Ca2+ sensing ability 53-55. Reasons for this downregulation is hypothetical and no somatic or germline mutation in the CaR gene have been found in sporadic HPT 56, 57. However, allelic loss for markers flanking the CaR gene has been detected in tumors in a small number of patients with primary HPT 58. General characteristics of vitamin D receptor (VDR) expression in parathyroid pathologies resemble those of CaR. Protein and mRNA levels are reduced and hitherto no mutation has been demonstrable in either primary or secondary HPT 59, 60. The most prominent decrease in VDR is seen in large glands and nodular hyperplasias of secondary HPT. This substantiates that the antiproliferative actions of active vitamin D requires VDR 61. The gene coding for LRP2 maps to chromosome 2q21-q22 and allelic loss has not been detected in parathyroid tumors analyzed 62. However, reduced expression of LRP2 has been demonstrated in sporadic parathyroid adenomas with respect both to protein and mRNA levels 63-65. The concomitant down-regulation of LRP2 and CaR has been suggested to be a molecular requisite for the right-shifted doseresponse curve between Ca2+ and PTH in diffuse and nodular hyperplasia as well as in adenomas 62, 63.. 11.

(19) SECONDARY HYPERPARATHYROIDISM. Secondary HPT develops in patients with chronic renal failure but can occasionally also occur due to other diseases and medications. In renal diseases there is longterm stimulation of the parathyroid glands due to decreased production of active vitamin D and hyperphosphatemia, while hypocalcemia may be temporary. In advanced stages there is considerable enlargement of the nodularly arranged parathyroid glands and markedly increased PTH secretion coexisting with hypercalcemia 66-68 . This growth cause an increased susceptibility to additional genetic hits and indeed, using X chromosome analysis 58-64% of the investigated patients with uremic HPT had at least one monoclonal gland 42, 69. Treatment with active vitamin D can suppress the excessive PTH secretion and reduce the proliferation rate 70, 71. The success of this treatment is usually less evident in advanced secondary HPT, which is consistent with the generally low VDR expression in large hyperplastic nodules 59, 60 .. FAMILIAL HYPERPARATHYROIDISM. MULTIPLE ENDOCRINE NEOPLASIA Multiple endocrine neoplasia (MEN) type 1 is an autosomal dominant tumor syndrome with over 95% penetrance and equal sex distribution 72. The syndrome is characterised by neoplasia of the parathyroid glands (90-97% of patients), the endocrine pancreas and duodenum (30-80%) and the anterior pituitary gland (15-50%) 73, 74. The patients also may develop lipomas, bronchial and thymic carcinoids, thyroid nodules, angiofibroma, adrenal lesions and spinal ependymoma 75-78. Some of these features, which earlier were considered uncommon, have been found in a significant proportion of the patients 79, 80. The disease locus is mapped to chromosome 11q13 and the gene (MEN1) encodes the tumor supressor menin 81, 82. Mutations have been identified in all coding exons of the gene and in more than 90% of the MEN type 1 families, but no clear genotype-phenotype correlation has been established 83-87. Hypercalcemia in MEN type. 12.

(20) 1 patients usually is mild, asymptomatic and detected through biochemical screening. Multiglandular parathyroid disease with marked phenotypic heterogeneity is the characteristic finding at operation 73. Parathyroidectomy involves a substantial risk of recurrence of HPT and is performed as radical subtotal resection leaving a 30-40 mg remnant in situ or as total parathyroidectomy with autotransplantation 88. Mutations in the RET proto-oncogene on chromosome 10q11 cause MEN type 2A, MEN type 2B and familial medullary thyroid carcinoma. MEN 2A is an autosomal dominant syndrome that includes medullary thyroid carcinoma, pheochromocytoma and HPT 89-91. HPT is seen in less than 30% of the MEN 2A patients 92, 93, and is rare in MEN 2B 92, 93. THE HYPERPARATHYROIDISM – JAW TUMOR SYNDROME Besides HPT the hyperparathyroidism-jaw tumour syndrome (HPT-JT) is associated with Wilm´s tumour, ossifying jaw fibromas, renal hamartomas, kidney cysts and polycystic kidney disease 94-97. HPT-JT has a sex dependent penetrance with a male predominance. HPT in these patients is associated with markedly elevated serum calcium and PTH levels. Histopathology often display large adenoma and a majority of the patients are cured by operative removal of a single enlarged parathyroid gland 94. There is, however, an increased incidence of parathyroid carcinoma. The syndrome has been mapped to 1q25-q31 (HRPT2-locus) 47, 98. LOH in this region is particulary seen in renal hamartomas and parathyroid carcinomas from HPT-JT patients, suggesting that the gene, yet to be characterised is a tumor suppressor. However, parathyroid adenomas more often show allelic retention 94, 96. FAMILIAL ISOLATED HYPERPARATHYROIDISM Familial isolated hyperparathyroidism (FIHPT) is defined as familial primary HPT without an overtly increased risk of other tumors. Furthermore, patients with FIHPT have been suggested to be at risk for parathyroid carcinoma 95, 99. FIHPT has been. 13.

(21) mapped to both the MEN 1 gene and HPT-JT locus 95, 100-102. Indeed mutation data of MEN type 1 still is inconclusive with occurrence of families without recognizable MEN 1 gene mutations 83, 85, 101, 102. Thus, whether FIHPT ia a variant of either MEN type 1 or HPT-JT or is a genetically distinct entity remains to be elucidated.. FAMILIAL HYPOCALCIURIC HYPERCALCEMIA. Familial hypocalciuric hypercalcemia (FHH) is an autosomal dominant condition with almost complete penetrance 103. Genetically, the majority of cases has been linked to the CaR gene on chromosome 3q with mutational abnormalities occurring preferably in extracellular and transmembrane coding regions of the gene 104. A small number of families with clinically similar phenotype have been linked to one of two loci on chromosome 19 (19p13.3, 19q13) 105, 106. Hypothetically, FHH in these families should relate to mutational aberrations in genes coding for proteins interacting with CaR signalling and/or expression. The hypercalcemia of FHH debuts at an early age and is mild-to moderate. Hypercalcemic members are generally considered to be asymptomatic, but significantly more mental problems, headache, fatique, weakness and arthralgia has been reported 103, 107, 108 . Pancreatitis has been described in some kindreds, although it may be no true complication of FHH as only sporadic cases has been seen in the families 103, 108-110. Markers of bone turnover may be mildly elevated, but bone mineral density is generally normal in affected members 103. Biochemical findings in affected individuals characteristically include mild hypercalcemia, low urinary calcium excretion, mild hypermagnesemia or serum magnesium levels in the upper part of the normal range, and normal levels of PTH, phosphorus and active vitamin D 103, 107, 108. There is a positive correlation between the serum total calcium and magnesium levels, which contrasts to the tendency of an inverse relationship in primary HPT 103. A characteristic finding in most persons with FHH is relative hypocalciuria 103, 107, 108. Approximately 80% of persons with FHH have a calcium to. 14.

(22) creatinine clearance ratio of less than 0.01, whereas at least a corresponding proportion of patients with primary HPT have a clearance ratio above this value 103. (Figure 1.). 0 .4 0 0. Primary HPT. FHH. 0 .2 00. 0 .1 00 0 .0 6 0 0 .0 4 0 0 .0 2 0. 0 .0 1 0. Figure 1 . Renal calcium to creatinine clearence ratio in patients with sporadic primary hyperparathyroidism or familial hypocalciuric hypercalcemia (FHH). Each point represents a different patient. (From: Marx SJ, Ann Int Med, 1980). 0 .0 0 6 0 .0 0 4 0 .0 0 2. °° °°°°°°°° °°° °°°°°°° ° °. °° ° ° ° °. 0 .0 0 1. Attempts to normalise the hypercalcemia of FHH by parathyroidectomy have been accompanied by unacceptable postoperative risks of both persistent hypercalcemia and hypoparathyroidism 103. Relative hypocalciuria persists after parathyroidectomy and is therefore considered to represent excessive renal re-absorption of Ca2+ in the thick ascending limb 111, 112 . The majority of patients with FHH have parathyroid glands that are indistinguishable from normal by size, weight and microscopic appearance. About 20% of the patients have slightly enlarged parathyroid glands when 75 mg were taken as normal upper limit 113. Whether or not these slightly enlarged glands represent hyperplasia is unclear.. 15.

(23) CALCIUM RECEPTOR MUTATIONS About 40 different CaR mutations have been identified in families with FHH 114-118. (Figure 2.) The majority is heterozygous missense mutations, but there are also nonsense mutations, deletions and insertions. Point mutations have not been found in the intracellular part of the CaR, although an inserted Alurepetitive sequence with predicted truncation of the protein has been shown in one family 116. The mutations lead to a highly variable increment of the set point of the PTH secretion in the parathyroid glands that may result from altered Ca2+ affinity, disturbed ability to mediate agonist binding of the mutated receptor, and decreased cell surface expression of the wild type CaR 119-123. In a mouse model for FHH with heterozygous inactivation of the CaR gene, the animals exhibited modest elevations of serum calcium, magnesium and parathyroid hormone levels as well as hypocalciuria and approximately 50% reduction of CaR expression in the parathyroid and kidney 124. Consequences of the reduced expression can be further aggravated by a dominant negative interaction of the mutant CaR through heterodimerization with the remaining normal receptors 125. Some inactivating CaR mutations, mainly homozygous but also heterozygous, can give rise to severe symptomatic hypercalcemia and markedly elevated PTH levels within the first week of life. (Figure 2.) The life-threatning symptoms include hypotonia, failure to thrive, constipation and respiratory distress and have been denoted ´neonatal severe hyperparathyroidism´ 107, 126, 127 . The condition is lethal unless the hypercalcemia is reversed and operatively removed parathyroid glands have all been markedly enlarged as also seen in a mouse model with homozygously inactivated CaR 124, 128. Activating mutations of the CaR gene have been found to cause hypocalcemia and relative hypercalciuria with normal PTH levels 129. However, mutations in the CaR have also been found in individuals with sporadic or familial hypoparathyroidism accompanied by both hypocalcemia and low PTH levels 130, 131. 16.

(24) Figure 2. Predicted topology of the calcium receptor (CaR) cloned from human parathyroid. Missense and nonsense mutations that cause familial hypocalciuruc hypercalcemia (FHH) or autosomal dominant hypercalcemia are indicated. These are represented using the three-letter amino acid code. The normal amino acid is shown before and the mutated amino acid is indicated after the nuber of the relevant codons. (Modified from Brown EM, Am J Med, 1999). 17.

(25) PARATHYROID TUMOR GENETICS IN SPORADIC HYPERPARATHYROIDISM. DNA damage in tumor suppressor genes and proto-oncogenes contributes to the development of neoplasia. Proto-oncogenes are often involved in the control of cellular growth, proliferation or differentiation, whereas tumor suppressor genes normally restrain cellular proliferation. The only proto oncogene with unequivocal involvement in parathyroid neoplasia is the Cyclin D1/PRAD 1 gene (parathyroid adenomatosis gene –1) on chromosome 11. About 5% of the parathyroid adenomas show pericentromeric inversion that brings the PRAD 1 gene under control of the PTH gene promoter region and leads to overexpression of PRAD 1 132. PRAD 1 encodes for cyclin D1 that plays an important role in regulation of cell cycle progression by controlling G1 to S cell cycle transition 133-136. Even if the PRAD 1 rearrangement only occurs in a subset of adenomas, PRAD 1 protein is overexpressed in 18% to 40% of adenomas and to some extent also in carcinomas and hyperplasias 137-141. Transgenic mice overexpressing cyclin D1 develop primary HPT 142 . Rearrangement and/or overexpression of PRAD 1 have also been detected in other neoplasias as b-cell lymphomas, squamous cancer of various tissues and breast tumors 143-146. Inactivation of both alleles of a tumour suppressor gene, by mutation or deletion, is required to completely deplete the gene´s antineoplastic product. A common inactivation mechanism is somatic deletion of a substantial portion of chromosomal DNA that includes the relevant gene. This can be revealed as loss of heterozygosity of DNA markers in tumor DNA relative to normal DNA of the same individual. Over 70% of the parathyroid adenomas have at least one such clonal defect. Allelic loss on chromosome 11q13 including the MEN 1 locus occurs in a majority of parathyroid tumors from MEN 1 patients, in a third of tumors of sporadic primary HPT and in some enlarged glands from patients with uremic HPT 147-150. Furthermore, about 50% of the sporadic primary tumors with. 18.

(26) allelic loss on 11q13 have also a somatic MEN 1 gene mutation 149-152 . LOH has also been detected on chromosome arms 1p, 6q, 11p, 15q in more than 25% of informative parathyroid adenomas, which emphasize the molecular heterogeneity of these neoplasias 153-157. The most common defect together with LOH on 11q13 is allelic loss on chromosome 1p36, which is found in over 40% of the adenomas 153. Interestingly, LOH on chromosome 1p occurs in several other neoplasms as medullary thyroid carcinoma and pheocromocytoma 158. Although several candidate genes are present in this region, the involvement in the development of parathyroid or other types of tumors remains to be established. Investigation of the retinoblastoma (Rb) gene on chromosome 13 confirmed allelic loss in all parathyroid carcinomas of 5 informative patients and in up to 20% of the parathyroid adenomas 159, 160. Allelic loss correlated with absence of immunostaining for the Rb protein, whereby it was speculated that Rb involvement might improve the sometimes difficult distinction between malignant and benign parathyroid neoplasia. Subsequent studies confirmed that Rb loss may couple to a clinically aggressive type of parathyroid tumors 160, 161. Comparative genomic hybridisation in which the entire tumor genome is screened for gains and/or losses, has confirmed the chromosomal losses detected by LOH and identified amplified regions on several chromosomes. Most consistently 7, 16p, 19p have been suggested to harbour putative parathyroid oncogenes 162-164 .. 19.

(27) AIMS OF THE STUDY The study was initiated by a middle-age male displaying hypercalemia, hypercalciuria and history of kidney stones, preliminary interpreted as primary HPT. Persistent hypercalcemia was reversed by parathyroid reoperation involving autotransplantation to the forearm of the only remaing parathyroid gland. The extraordinary circumstance was that this gland was normal in size. This prompted re-interview of the patient and some of his relatives, whereby familial hypercalcemia with rather dismal outcome after primary parathyroidectomy procedures were unveiled. These findings prompted further exploration of the family. The purpose of this studies was to clinically and molecularly investigate the family with hypercalcemia with special respect to: - defining the mode of inheritance, mapping the disease locus and identifying the causing genetic abnormality - clinical characteristics in the affected family members - possibilities for treatment by parathyroidectomy - genome wide study for LOH in the parathroid glands - mRNA expression of functionally important genes in the parathyroid tumors. 20.

(28) MATERIAL & METHODS PERSONS & TUMORS (Paper I-VI) Screening for hypercalcemia in the family revealed more than 200 members, and 22 affected (hypercalcemic) and 56 unaffected (normocalcemic) members were scrutinized further (Paper I). Figure 3. Parts of the kindred remain to be investigated and this includes all individuals under the age of 18 years. Altogether 17 affected family members, 5 women and 12 men, underwent parathyroidectomy during 1983 – 1999 (Paper II). Postoperative follow-up is 5.1±1.2 years. This subgroup showed no significant difference from all the identified family members with hypercalcemia, but for a somewhat younger age. Nine parathyroid glands from eight parathroidectomised family members were investigated more closely concerning allelic loss and mRNA levels (Paper III, IV). Normal and abnormal parathyroid tissue for comparison was obtained from 12 patients with single adenoma, 12 patients with HPT secondary to renal insufficiency and eight histopathologically normal glands from patients with single adenoma of primary HPT (Paper IV). Histopathologically normal parathyroid cells from 15 patients operated for atoxic goitre were used as reference in Paper I. BIOCHEMISTY IN BLOOD AND URINE (Paper I-IV) Biochemical analyses utilised our routine clinical laboratory. Determinations of urinary calcium, creatinine and phosphorous excretions were performed using 24-hour collections. The calcium to creatinine clearance (Ca/CrCl) ratio was calculated as (s-Ca x u-Cr / u-Ca x s-Cr) as described 103. The ionized calcium concentration corresponding to 50% inhibition of the maximal PTH secretion (ED50) was determined in vivo by induction of hypo- and hypercalcemia by sequential infusion of gradually increasing concentrations of sodium citrate, followed by calcium chloride in six healthy volunteers and in six family members before and after parathyroidectomy as described 52, 165.. 21.

(29) Figure 3. Pedigree of the family. Males are represented with squares; women with cirkles and slashes indicate deceased individuals. All the 22 identified hypercalcemic members (filled symbols) are shown but not all of the 56 normocalcemic ones (grey) symbols. Open symbols indicate that the calcium levels are unknown.. PARATHYROID HISTOLOGY (Paper I-IV) Adenoma was defined as a single enlarged parathyroid gland with increased mass of parathyroid cells from a patient with HPT. Parathyroid hyperplasia, albeit pathogenetically equivocal, was defined as enlargement of two or more parathyroid glands with increased mass of parenchymal cells. Identification of hyperplastic nodules required reasonably well-defined groups of morphologically similar cells with contrasting phenotype to the surrounding parenchyma. IMMUNOSTAINING (Paper II) Cryosections (6µm) of parathyroid glands from affected family members were fixed in acetone and stained with a mouse monoclonal antibody (E11) that recognizes the cell surface. 22.

(30) protein LRP2 in normal parathyroid, placenta, type 2 pneumocytes and proximal kidney tubular cells 22, 166, 167. The production and characterisation of the antibody has been described 168. The immunostaining was performed with a mouse peroxidase-antiperoxidase (PAP) technique and the sections were counterstained with Mayer's hematoxylin 168. DNA & RNA ISOLATION AND cDNA SYNTHESIS (Paper I, III-IV) Genomic DNA was prepared from whole blood using standard methods or genomic DNA purification KIT (Wizard, Promega, Madison, WI) 169. Operatively removed parathyroid tissue was immediately frozen in liquid nitrogen and stored at -70°C. Genomic DNA and total RNA from parathyroid specimens was isolated using TRIZOL reagent (Gibco, Life Technologies, Grand Island, NY) or QIAGEN RNA/DNA kit (Qiagen, Hilden, Germany). The RNA samples were treated with RQ1 Rnase-Free Dnase (Promega, Madison, WI) to avoid contamination of genomic DNA. First strand cDNA was prepared from total RNA using First Strand cDNA Synthesis Kit (Amersham Pharmacia Biotech Inc., Buckinghamshire, England). DNA was quantified spectrophotometrically and quality assessed on a agarose gel. SEQUENCE ANALYSIS (Paper I) Mutational analysis was performed by semiautomated sequence analysis using ABI PRISM Dye Terminator cycle sequencing ready reaction kit with AmpliTaq DNA polymerase (PE Applied Biosystems, Foster City, CA) after PCR amplification. Exons 2, 4-7 of the CaR gene was sequenced using primers 1F-1R, 2F-2R, 3AF3AR, 3BF-3BR, 4F-4R, 5F-5R, 7GF-6AR, 6BF-7ER, 7FF-6BR, 6CF6CR and 6DF-6DR (KEBOLab, Stockholm, Sweden) 104, 118 in four hypercalcemic and four normocalcemic individuals of the family, and finally in all more closely investigated family members with use of primers 7TCF (5´-ggatctccttcattccagcctatgc-3´) and 7TCR (5´-gggctgctgctgagatcgttgctgc-3´) (Paper I). Exons 2-7 were also sequenced in both parathyroid tissue and leucocytes from four affected family members (Paper III). Both DNA strands of the amplified segments were analysed.. 23.

(31) SITE-DIRECTED MUTAGENESIS AND TRANSIENT RECEPTOR EXPRESSION (Paper I) Site-directed mutagenesis was performed to produce a receptor containing the identified point mutation (F881L) in the CaR gene of the affected family members 170. Incorporation of the desired mutation was confirmed by sequencing. A vector containing wild-type or mutated CaR was transfected into HEK 293 cells (provided by NPS Pharmaceuticals, Inc., Salt Lake City, UT) using LipofectAMINE (Gibco BRL, Gaithersburg, MD) as a DNA carrier. MEASUREMENT OF INTRACELLULAR CALCIUM (Paper I, II) Forty-eight hours after transfection coverlips with attached HEK 293 cells were loaded for two hours with FURA-2/AM (Calcbiochem, La Jolla, CA) in 20 mM HEPES at room temperature. To measure intracellular Ca2+ in the cells the coverslips were put in a cuvette in which extracellular Ca2+ was stepwise increased. Individual cells were examined in an inverted microscope equipped with a perifusion chamber allowing rapid changes in the extracellular Ca2+ concentration. Emitted fluorescence from individual cells was measured at 510 nm and the ratio of emission at 340/380 nm excitation was used to calculate intracellular Ca2+ in the different parathyroid cells 121. Parathyroid cells were generated from 10 parathyroid glands from four affected family members and cells from normal glands of 15 patients operated for atoxic goitre and loaded with Fura/AM as described 171. Measurement of intracellular Ca2+ in parathyroid cells was performed as above.. 24.

(32) LOSS OF HETEROZYGOSITY (Paper III) Genomic DNA from the nine parathyroid lesions and paired leucocyte DNA were subjected to PCR amplification with highly polymorphic microsatellite markers throughout the human genome. 139 primers from Weber Set 6 (kindly provided by Nordic Consortium Primer Resource Center, Dept of Genetics and Pathology, Uppsala University Hospital) and version 9a of the Human Screening Set-ABI Dyes (Genetic Research, Huntsville, AL) were used. Informative results were obtained from all chromosomal arms except the short arms of the acrocentric chromosomes. The fluorescent-labelled PCR products were quantified with GeneScan Software on an ABI 310 semi-automated sequencer with GeneScan TAMRA 350 as size marker. Chromosomal arms 1p, 1q, 6q, 11q and 15q were analysed more closely using both fluorescent and 32P labelled primers. When using 32P-labeled markers, the PCR products were quantified on a PhosphorImager. The level of allele retention for each microsatellite marker was calculated by comparing the relative signal intensity of the two alleles in the tumor with that of the two alleles in the normal tissue (T1/T2)/(N1/N2). Allelic loss was defined as a reduction of 50% or more in the comparison. (Figure 4.). Figure 4. Example of different LOH categories. (A). Retention of microsatellite marker D3S1269 and D15S822 on chromosome 3q and 15 resectively. (B). Example of allelic loss (37%) of marker D12S1045 on cromosome 12q.. 25.

(33) REAL-TIME QUANTITATIVE PCR (Paper IV) Relative RNA expression of CaR, VDR, PTH, LRP2 and a proliferation marker, PCNA, was determined by real-time quantitative PCR (TaqMan) in nine parathyroid glands from 8 of the affected family members 172. Parathyroid tissue from 12 patients with single adenoma, 12 patients with HPT secondary to renal insufficiency and 8 histopathologically normal glands from patients with single adenoma of primary HPT were analyzed for comparison. Gene-specific primers and flourogenic probes were designed using PrimerExpress (PerkinElmer, Foster City, CA). Each probe has a fluorescent reporter dye and a quencher dye at the 5’ and 3’ end, respectively. The quencher inhibits reporter dye emission in an intact probe. During the PCR extension phase the annealed probe is cleaved by the 5’ to 3’ exonuclease activity of Taq DNA polymerase. The cleavage produces an increase of reporter dye fluorescence emission, which is proportional to the target gene expression in the investigated sample. Relative quantity was measured against a standard curve generated from dilution series of target specific PCR fragments for the investigated genes. The reactions were performed and analysed using Applied Biosystems PRISM 7700 Sequence Detector (PE, Foster City, CA). Each sample was analysed in triplicates and normalised to glyceraldehyde-3-phosphate dehydrogenase (GAPDH). All TaqMan assay reagents were obtained from PerkinElmer applied Biosystems. LINKAGE ANALYSIS (Paper I) Linkage analysis was performed by amplifying leucocyte DNA from family members with specific primers from 1p, 1q, 2q, 3q, 11q and 19p. A potential parathyroid tumor suppressor gene is located to 1p, 1q is the locus of HRPT-2, 2q that of the LRP2 gene, 3q harbours the CaR gene, 11q the MEN 1 gene, and one FHH family has been mapped 19p 47, 81, 106, 153, 173, 174. After amplification by PCR, the reactions were run on a denaturing 4.5% acrylamide sequencing gel and visualised by autoradiography so the genotypes could be determined. Twopoint linkage analysis was performed using the FASTLINK. 26.

(34) program, assuming various penetrance frequencies 175. Conventional levels for the lod scores calculated were used i.e. a lod score >3.0 signifies linkage (p<0.01) to a given marker and that of >-2.0 excludes linkage. STATISTICAL ANALYSIS (Paper I-IV) Wilcoxon non-parametric rank sum test, Student´s paired and unpaired two-tailed t-test, ANOVA and Pearson's correlation coefficients were used for statistical analysis. All results are expressed as mean±SEM and p<0.05 was considered significant.. 27.

(35) RESULTS & DISCUSSION. MUTATIONAL CHARACTERISTICS (Paper I) Screening revealed autosomal dominant inherited hypercalcemia in 22 affected (hypercalcemic) and 56 unaffected (normocalcemic) members. The hypercalcemia was linked to the CaR gene locus on chromosome 3q between markers D3S1303 and D3S1269 with maximum LOD score of 4.25 and 5.39, respectively. Sequencing of exons 2-4 and 7 of the CaR gene demonstrated a previously unknown heterozygous T to C transition in exon 7 that results in a phenylalanine to leucine substitution in the intracellular part of the CaR gene. Subsequently, this mutation was identified in all affected family members, but not in any of the normocalcemic members. Automated sequencing of 100 healthy Caucasians did not identify this sequence variant, suggesting that is does not represent a common polymorphism 176. Inactivating mutations in the CaR gene causing FHH have only been seen in the extracellular or transmembrane domain of the CaR gene 177. Mutation in the extracellular part likely effect the ligand binding, while this novel receptor might affect the intracellular responses more directly 177, 178. Causal coupling of the mutation to the hypercalcemia has been substantiated by the action of extracellular Ca2+ on the intracellular Ca2+ concentration in HEK 293 cells transiently transfected with the mutated (F881L) and wild-type receptor in vitro. The mutant receptor showed a significantly right-shifted dose-response curve (p<0.05) compared with the wild-type receptor. EC50 for the two variants were 4.9±0.1 mM and 4.1±0.1 mM, respectively. The extracellular Ca2+ regulation of intracellular Ca2+ in dispersed parathyroid cells from 10 family members showed increased EC50 similar to that seen in cells from parathyroid adenomas 179. These results is consistent with inactivation of the receptor as seen in FHH. However, FHH families with similar degree of hypercalcemia as the described family have displayed more pronounced increase of EC50 upon functional analyses of the mutant receptor 177, 178 .. 28.

(36) CLINICAL CHARACTERISTICS (Paper I, II) The affected individuals displayed moderate hypercalcemia, inappropriate high intact serum PTH levels and mild hypercalciuria. Two hypercalcemic individuals provided a history of renal stones. Ten more extensively studied family members had normal creatinine clearance together with significantly higher urinary calcium excretion compared with 11 non-affected relatives. The calcium-creatinine clearance ratio (Ca/CrCl) was raised above the diagnostic level of FHH (0.010) in 7 out of 10 affected family members. Screening for the endocrine pancreatic and pituitary involvements of MEN 1 and jaw tumors was negative 73. The affected family members also displayed inappropriate high levels of intact PTH and relative hypermagnesemia. (Table 1.) Serum calcium was lowered to normal levels (range 2.22-2.56 mM) by parathyroidectomy in all 12 radically parathyroidectomized patients (see below). However, it remains to be evaluated whether the normocalcemia will persists for decades. Intact PTH and 24-hour calcium excretion were also significantly lowered by the operation, while serum phosphate and magnesium increased. (Table 2.) Dynamic evaluation of parathyroid function before surgery showed baseline serum ionized calcium and intact PTH of 1.34 mM and 32.2 ng/l, respectively. The corresponding postoperative values were 1.17 mM and 22.4 ng/l. ED50 was reduced from 1.31±0.04 mM to 1.17±0.05mM (p<0.05). Six healthy volunteers had an average ED50 of 1.15±0.004 mM which was very similar to that of the members having undergone apparently successful parathyroidectomy. Biochemically there are signs consistent with FHH in this family. These signs include mild to moderate hypercalcemia and relative hypermagnesemia 103. The similarity with FHH also includes the point mutation in the CaR, since decreased expression but not genomic alterations have been found in HPT. On the other hand, relative hypercalciuria, inappropriately elevated serum PTH and relatively high calcium-creatinine clearance ratio mimic HPT and contrast to the typical findings in FHH 180. The findings. 29.

(37) substantiate that the previously unrecognised point mutation in the intracellular domain of CaR in the family couples to a novel variant of hypercalcemia that can be an intermediate variant of FHH and HPT. Prevalence of this variant is unknown, and it also remains to be determined if it may contribute to postoperatively persistent hypercalcemia in mainly young to middle-aged individuals with modereate degree of hypercalcemia. Table 1. Characteristics (mean±SEM) of affected (hypercalcemic)and nonaffected (normocalcemic) members of the family.. Age, year s-Calcium, mM Ionised pcalcium, mM Intact s-PTH, ng/L s-Magnesium, mM s-Phosphate, mM s-creatinine, µM dU-Calcium, mmol/24h Ca/CrCl. Non-affected. Affected. P. Reference range. n=17-22 47.6±2.3 2.43±0.01. n=10-56 48.6±2.3 2.82±0.02. n.s. <0.0001. n.a. 2.20-2.60. n.i.. 1.35±0.02. 32±2. 40±2. <0.05. 12-55. 0.82±0.009. 0.87±0.009. <0.05. 0.70-0.91. n.i.. 1.01±0.06. 86±3. 89±2. n.s.. 64-106. 3.1±0.4. 6.1±1.0. <0.05. 0.5-5.0. 0.0079±0.001. 0.012±0.002. <0.05. <0.010. 1.10-1.30. 0.74-1.54. S; serum, PTH; parathyroid hormone, n.s.; not significant, n.a. not applicable, n.i.; not investigated. 30.

(38) Table 2. Characteristics (mean±SEM) of the subgroup of parathyroidectomized family members before and after operation.. Before surgery. After surgery. P. n=9-17. Reference. range. n=9-17. Age, year s-Calcium, mM Ionised pcalcium, mM Intact s-PTH, ng/L s-Magnesium, mM s-Phosphate, mM s-creatinine, µM dU-Calcium, mmol/24h dU-Magnesium, mmol/ 24h dU-Phosphate, mmol/24h Ca/CrCl. 43.6±2.6 2.83±0.02 1.35±0.02. 48.2±3.8 2.46±0.04 1.17±0.04. n.a. n.a. <0.001 2.20-2.60 <0.05 1.10-1.30. 42±2. 31±4. <0.05. 12-55. 0.85±0.02. 0.90±0.02. <0.05. 0.70-0.91. 0.93±0.06. 1.14±0.08. <0.05. 0.74-1.54. 90±2 6.4±1.0. 96±4 3.1±0.6. n.s. <0.05. 64-106 0.5-5.0. 5.2±0.6. 4.5±0.5. n.s.. 2.5-7.5. 35±3. 32±3. n.s.. <38.4. 0.012±0.002. 0.0085±0.003. n.s.. <0.010‡. S; serum, p; plasma, dU; 24h-urine, PTH; parathyroid hormone n.a.; not applicable, n.s.; not significant ‡ Estimated upper limit of familial hypocalciuric hypercalcemia. 31.

(39) PARATHYROIDECTOMY PROCEDURE (Paper II) In the index case (middle-aged male), hypercalcemia persisted post-operatively despite that only one normal-sized parathyroid gland out of four identified, was left in situ. Two of the excised glands showed diffuse hyperplasia and the third was considered to be normal. Re-operation two years later because of gradually increased serum calcium levels, led to normocalcemia after autotransplantation of the single parathyroid gland with mild diffuse hyperplasia. Subsequently, two women were parathyroidectomized with histopathological finding of double adenoma and four gland hyperplasia with partial nodularity, respectively. The first of the two women has remained postoperatively normocalcemic, while the other woman exhibited an estimated parathyroid remnant of 75-85 mg has persistent hypercalcemia. After recognition of familial hypercalcemia an increasingly radical surgical procedure was attempted to reach postoperative normocalcemia. Subsequently, parathyroid surgery was performed in 5 women and 12 men of the affected family members. The first five members underwent parathyroidectomy for supposedly sporadic HPT with excision of only the enlarged glands or subtotal parathyroidectomy. The remaining 12 individuals were radically parathyroidectomised with the intent to leave a remnant of less than a normal-sized gland in the neck (ie. 10-20 mg from the smallest gland of each individual). Operative results improved dramatically with this procedure. Postoperatively persistent hypercalcemia thus was recorded in 3/5 individuals prior to the change in strategy and in 0/12 when the final procedure had been implemented. The nessessary radicality of resections differs from both sporadic HPT and parathyroid tumor susceptibility syndromes 73, 181. In FHH attempts of parathyoidectomy generally have led to persistent hypercalcemia or hypoparathyoidism 103. Histopathologically, the glands consisted of diffuse chief cell hyperplasia in 12 individuals, while nodular formations occurred in one or two glands from the other five, which initially were misinterpreted as adenomas. FHH is associated with normal or. 32.

(40) mildly hyperplastic parathyroid glands and rarely have nodular changes. Total weight of the excised parathyroid tissue increased with age of the patients (r2=0.44, p<0.05) and averaged 394±50 mg. This substantiates a mildly elevated cell proliferation. 29% of the operated family members had parathyroid glands in atypical positions which exceed the findings in autopsy and operative materials of primary HPT 29, 36. Immunochemical LRP2 staining showed maintained expression in glands with diffuse hyperplasia, while nodular gland parts had reduced immunostaining. These findings are consistent with the staining for LRP2 in sporadic HPT 63. To conclude, the hypercalcemia was reversed with unusually radical parathyroidectomy. More substantial parathyroid remnants tend to be associated with a higher frequence of persistent hypercalcemia, while, recurrence of hypercalcemia has not yet been recorded in those more radically operated. Nevertheless the duration of follow-up in the more optimally operated individuals (3.1±0.3 years) is similar to what generally is regarded as a sign of cure in sporadic primary HPT. The parathyroid glands displayed diffuse to nodular hyperplasia and the generally mild increase in size apparently progressed with age.. Figure 5. Schematic location of the upper (closed symbols) and lower (striped symbols) parathyroid glands with abnormal location in 5 family members. Number refers to Figure 3.. 33.

(41) LOSS OF HETEROZYGOSITY (Paper III) Informative results were obtained from all chromosomal arms of the nine examined tumors with exception for the acrocentric ones by use of the 139 microsatellite markers. Each parathyroid gland had allelic loss on at least one chromosomal arm and the highest frequency was seen in the gland with a very large, dominating nodulus. The frequency of allelic loss correlated to age of the patients at operation (p<0.01, r2=0.66), but not to the preoperative degree of biochemical derangements. Adenomas of primary HPT have shown monoclonality with allelic loss on particular 1p, 1q, 6q, 11q and 15q, whereas glands from FHH remain to be investigated in this respect . In this study, allelic loss was found on 12q in 56%, 6q and 7q in 44% each, 15q and 17q in 33% each, and on 1p, 7p, 10p and 19q in 22% each. The frequency of LOH on 1p, 6q and 15q was similar to that of non-familial primary HPT, whereas LOH on 12q, 7q and 17q were more frequent . In about 25-40% of the tumors of spordic HPT, allelic loss have been found on 11q13 and somatic mutations in the MEN 1 gene occur in approximately 50% of them . In almost all tumors from MEN type 1 families, allelic loss on 11q13 have been demonstrated. In contrast the family showed allelic loss on 11q13 very sparsely. The results indicate minor importance of the MEN 1 gene in the tumorgenesis of this family. No allelic loss was found on chromosome 13 where the Rb gene is located. LOH of the Rb locus has been found in about 20% of sporadic adenomas and in higher frequency in parathyroid carcinomas . Allelic loss on 12q was found in as much as 56% of the familie‘s tumors, which contrast to findings in other kinds of parahyroid tumors. Although no characterised tumor suppressor genes are located at this locus, LOH on 12q has been found in several other neoplasias such as those arisumg in the gall bladder, ovary and exocrine pancreas . Two potential minimal deleted regions could be detected, one located between markers D12S824 and D12S81 and one telomeric of D12S821. Potential importance of these minimal deletion regions should not be overemphasized since the number of investigated tumors 153-157. 153, 155. 148-151, 182. 159, 160. 183-185. 34.

(42) is limited. Similar to parathyroid tumors of sporadic HPT, no allelic loss was detected on chromosome 3q using markers flanking the CaR gene, neither could any smaller somatic mutational abberations be found in the CaR gene . The abnormal parathyroid glands of this family displayed characteristics consistent with monoclonal lesions and allelic loss at some loci infrequently deleted in other parathyroid lesions such as 12q and 7q. Hypothetically, the germline CaR mutation of the present family cause polyclonal proliferation, making the parathyroid cells susceptible to other genetic hits. Indirect support for this hypothesis resides in the increased number of allelic losses and size of the parathyroid lesions with age of the affected family members. This would coincide with the present hypothesis of parathyroid tumor development in primary and secondary HPT . 57, 58. 186. TABLE 3. Summary of the screening for loss of heterozygosity in the nine investigated tumours. Chromosomal arms Tumour nr 1 2 3 4 5 6a 6b 7 8. 1 p q × × ♦ × × × × × ♦ × × × × × × × × ×. 2 p q × × × × × ♦ × × × × × × × × × × × ×. 3 p q × × × × ♦ × × × ♦ × × × × × × × × ×. 4 p q × × × × × × × × × × × × × × × × × ×. 5 p q ×× × × × × × × × × × × × × × × × ×. 6 p q × × × ♦ × ♦ × × × × × ♦ ×♦ × ® × × ×. 7 p q × × × ♦ ♦ × ♦♦ × ♦ × × × ♦ × × × ×. 8 9 p q p q × × × × × × × × × × × × × × × × × × × × × × × × × ×× × × × × × × × × ×. 10 p q × ♦ × × × × × × × × × × × × × × ♦ ×. 11 p q × ♦ × × × × × × × × × × ♦ × × × × ×. ♦, loss of heterozygosity; ×, retention of heterozygosity. 35. 12 131415 16 5 q p q p q q q × × × × × × × ×♦ × × ♦ × × ® × × × × × ×♦ × ♦ × × × × × × × × × × × × × ♦ × × ♦ × × ×♦ × ×♦ × × × ♦ × × × × × × × × × × × ×. 17 p q × × × × × ♦ × × × × × ♦ × ♦ × × × ×. 18 p q × × × × × × × × × × × × × × × × × ×. 19 p q × × × ♦ × × × × × × × × × ♦ × × × ×. 20 2122 p q q q × × × × × × × × × × × × × × × × × × × × × × × × × × × × × ♦ ♦ × × × × ×.

(43) QUANTIFICATION OF RELATIVE mRNA LEVELS (Paper IV) The PTH hypersecretion and increased parathyroid cell proliferation of primary and secondary HPT have been associated with down-regulation of messenger RNA and protein levels for CaR, LRP2 and VDR. The cause for down-regulation of CaR and the other functionally relevant proteins is unknown, but bovine parathyroid cells in monolayer culture show decreased CaR mRNA levels when loosing responsiveness of the PTH release to external Ca2+ . Similarly, LRP2 expression in several cell lines decreases with the degree of differentiation . In the present study, the glands of primary and secondary HPT showed the expected decrease in relative mRNA levels for CaR, VDR, PTH and LRP2 when compared to normal parathyroid glands. In the parathyroid lesions of the present family, CaR and VDR mRNA levels were reduced versus the normal ones. Significantly higher mRNA levels relative to GAPDH were noted for CaR and PTH when the glands of the family members were compared to primary or secondary HPT. A similar relationship was noted for LRP2 when compared to primary HPT. Increased proportion of hormonally active cells has been suggested to contribute to the PTH excess in HPT, since the mRNA and intracellular protein levels are decreased in primary and secondary HPT . Such compensation seems unlikely in the glands of the family as they showed apparently retained PTH mRNA levels. Lowered PTH content has been demonstrated in histologcally normal parathyroid glands from patients with HPT, ie. those accompanied by single parathyroid adenoma, when compared to glands of euparathyroid individuals . Consequently, the reference tissue of the present study can inaccurately reflect the euparathyroid situation. Interestingly, there was a trend for greater reduction in the VDR mRNA expression in nodular hyperplasia in the family, which coincides with findings in secondary HPT in which progression from diffuse to nodular hyperplasia seems to be accompanied by resistance to active vitamin D treatment and more pronounced derangement in calcium-regulated PTH secretion . 53. 187, 188. 51, 189. 190. 59. 36.

(44) Especially, the glands of secondary HPT had raised proliferation index (PCNA/GAPDH ratio), while the glands of the family members mimicked the normal reference. There was no correlation between PCNA mRNA ratios and weight of the glands in the family, which is quite consistent with the hypothesis of step-wise progression of HPT in sporadic cases. Summarizing, the rather mildly enlarged and hypercellular glands of the affected family members that underwent parathyroidectomy showed a relatively modest increase in an index of parathyroid proliferation, relatively mild decrease in CaR and VDR mRNA levels and generally retained LRP2 and PTH mRNA expression. It is noteworthy that the parathyroidectomized individuals naturally comprised a biased selection among the affected family members. There consequently are some members with hypercalcemia who fail to meet diagnostic criteria of HPT on the basis of too modest rise in serum PTH levels. There also are those who refuse parathyroidectomy, and yet others in which parathyroid tissue could were unavailable for analysis.. 37.

(45) GENERAL SUMMARY The studies reveal a hitherto unknown familial condition showing clinical characteristics of both FHH and HPT. The hypercalcemia was mapped to a point mutation in the intracellular part of the CaR gene that was functionally coupled to relative calcium resistance of the PTH release by transient transfection analysis. Apparent normalisation of the calcium homeostasis was induced by parathyroidectomy provided use of an unusually radical surgical strategy. At microscopic analysis, the glands displayed hyperplasia with nodular components in some of the patients. The removed glands were found to be monoclonal tumors with a high frequency of allelic loss especially on 12q, which contrasts to findings in sporadic or secondary HPT. Allelic loss was also seen in loci typical for primary HPT like 1p, 6q and 15q, but not 11q13. When compared to other parathyroid pathologies, the familial parathyroid glands showed a mild increase in proliferation rate and less reduced expression of genes critical for normal parathyroid cell function. The findings expand the current knowledge on supposedly rare variants of hypercalcemia that may be relevant also in the search for seemingly unexplainable causes for persistent hypercalcemia after apparently typical routine operations for HPT. There is also much more to learn about the present disorder. Issues that remain to be explored include the age at onset of hypercalcemia. Theoretically it may exist very early in the life of the mutation carries, but young family members remain to be investigated. Moreover, the examination of symptoms of the hypercalcemic family members has been rather simplistic. Besides medical history of renal calculi in a few individuals, most appear apparently asymptomatic. However, this is no objective measure on the extent of symptoms. There are in more detailed analyses of sporadic primary HPT findings of a more subtle depressive and anxiety- related symptomatology.. 38.

(46) ACKNOWLEDGEMENTS This work was performed at the Department of Surgical Sciences, Uppsala University Hospital, Sweden. Many people have contributed to this work and I would like to express my sincere gratitude to all of them and especially to: The investigated family and especially Torsten, with whom everything started, for the superb collaboration and understanding for our scientific work. Nothing of this could have been done without your fantastic contributions. Jonas Rastad, my main-supervisor, for his endless energy, encouragement and support, and for learning me everything I know about buying a car. Tobias Carling, thank you for being a competent supervisor and my teacher in laboratory work, “do it again, do it right”. Gunnar Westin, for co-authorship and helping me with laboratory techniques. Göran Åkerström for generous support throughout the work and guidance whenever needed. All co-authors, for fruitful collaboration and valuable opinions about the manuscripts. Special thanks to Ewa Lundgren who made me interested in research and for serving me wonderful cups of tea. Ola Hessman for helping me with allelic loss and initiating the majority of social activities of our laboratory group. Per Hellman for helping me to organize CICA. My laboratory group Birgitta Bondesson, Peter Lillhager, Ulrika Segersten, Per Hellman, Ola Hessman, Pamela Correa, Daniel. 39.

(47) Lindgren, Anders Knutsson, Peyman Björklund and Kenko Cupisti for being good lab-partners and friends. To my “every-day lunch-dates” Fredrik Stiger, Stina Johansson, Helena Brändström and Ulrika Segersten for helping me with laboratory/ technical problems and making my days a wonderful mixture of work, laughs, tea, planning social activities and deep discussion of life. Ulf Haglund, Head of the Department of Surgery and David Bergqvist, and Lars Viklund former and present Head of the Department of Surgical Sciences, for providing excellent conditions for research. Andreas Kindmark, Östen Ljunggren, Kenneth Jonsson, Håkan Melhus, Gisela Barbani , Håkan Hedstrand, Jan Melin and Peter Stålberg for the help in one way or another or just a chat in the corridor. My friends, close and distant, who fill my life with joy and who always have time to listen whenever I need. My collegues in Mora, for providing me a wonderful clinical education and giving me distance to my laboratory work, and Mia Andersson, Maria Lundekvam and Karin Bäckström, who make my time in Mora full of fun and adventures I have never dreamt of. Kalle, for being my very best friend. My family, my father Peter, my mother Monica and my sister Maria, for making me the one I am today and giving me endless love and support whatever I am doing.. 40.

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