Molecular Genetic Aspects of Sporadic Multiglandular Primary Hyperparathyroidism.

Multiglandular primary hyperparathyroidism (MGD) represents a rare form of primary hyperparathyroidism (PHPT). MGD is associated with hereditary PHPT, but the sporadic MGD is more common and affects a similar patient profile as single gland parathyroid disease (SGD). The distinction between SGD and MGD is of great clinical importance, especially for the strategy of parathyroidectomy. Based on the limited knowledge available, MGD is likely to be a genetically heterogeneous disease resulting from the interaction of germline and somatic DNA mutations together with epigenetic alterations. Furthermore, these events may combine and occur independently in parathyroid tumors within the same individual with MGD. Gene expression profiling has shown that SGD and MGD may represent distinct entities in parathyroid tumorigenesis. We are waiting for studies to analyze exactly which genes are different in SGD and MGD in order to identify potential biomarkers that can distinguish between the two forms of the disease.

Multiglandular Parathyroid Disease in Primary Hyperparathyroidism With Inconclusive Conventional Imaging.

Inconclusive preoperative imaging is a strong predictor of multiglandular parathyroid disease (MGD) in patients with primary hyperparathyroidism (PHPT). MGD was investigated in a cohort of 17 patients with PHPT (mean age 64.9 years, total calcium 2.75 mmol/l and parathyroid hormone (PTH) 113.3 ng/l) who underwent 18F-fluorocholine PET/CT (FCH) imaging before surgery. The initial MIBI SPECT scintigraphy (MIBI) and/or neck ultrasound were not conclusive or did not localize all pathological parathyroid glands, and PHPT persisted after surgery. Sporadic MGD was present in 4 of 17 patients with PHPT (24 %). In 3 of 4 patients with MGD, FCH correctly localized 6 pathological parathyroid glands and surgery was successful. Excised parathyroid glands were smaller (p <0.02) and often hyperplastic in MGD than in single gland disease. In two individuals with MGD, excision of a hyperplastic parathyroid gland led to a false positive decline in intraoperative PTH and/or postoperative serum calcium. Although in one patient it was associated with partial false negativity, parathyroid imaging with FCH seemed to be superior to neck ultrasound and/or MIBI scintigraphy in MGD.

Familial hypocalciuric hypercalcemia in an index male: grey zones of the differential diagnosis from primary hyperparathyroidism in a 13-year clinical follow up.

Familial hypocalciuric hypercalcemia (FHH) type 1, caused by a heterozygous inactivating mutation of the gene encoding the calcium-sensing receptor (CaSR), is characterized by mild to moderate hypercalcemia, hypocalciuria and inappropriately normal or elevated parathyroid hormone (PTH). FHH must be differentiated from primary hyperparathyroidism (PHPT) because parathyroidectomy is ineffective in the former. Herein, we report a 39-year-old male patient with a 13-year history of asymptomatic PTH-dependent hypercalcemia (mean calcium of 2.88 mmol/l; reference range 2.15-2.55 mmol/l) and calcium-to-creatinine clearance ratio (Ca/Cr) ranging from 0.007 to 0.0198, which is consistent with either FHH or PHPT. Although a family history of hypercalcemia was negative, and PET-CT with fluorocholine was suggestive of a parathyroid adenoma, genetic analysis of the CaSR gene identified a heterozygous inactivating mutation NM_000388.4:c.1670G>A p. (Gly557Glu) in exon 6 and a polymorphism NM_000388.4:c.1192G>A p. (Asp398Asn) in exon 4. The G557E mutation has been previously reported in a Japanese family in which all family members with the mutation had Ca/Cr below 0.01 consistent with FHH. The biochemical profile of FHH and PHPT may overlap. Our FHH patient with a G557E CaSR mutation illustrates that the differential diagnosis can be difficult in an index case with no family history, (false) positive parathyroid imaging and higher calciuria than expected for FHH. Calcium intake, vitamin D status and bone resorption might have contributed to the Ca/Cr variations over a 13-year clinical follow up. This case thus emphasizes the irreplaceable role of genetic testing of the CaSR gene when clinical evaluation is inconclusive.

Genetic predictors of the development and recurrence of Graves' disease.

Graves' disease affects approximately 3 % of women and 0.5 % of men. The first-choice therapy is based on the administration of thyrostatic drugs. However, approximately half of patients relapse within two years of discontinuation. These patients must then decide whether to re-initiate thyrostatics, which may have serious side effects, or to undergo surgery or radioiodine treatment. Familial forms of Graves' disease indicate a significant genetic component, with twin studies demonstrating a contribution of genetic factors up to 70-80 %. The autoimmune nature of the disease involves the human leukocyte antigen (HLA) complex, which has a decisive impact on each individual's immune response. Within HLA, some variants of the DRB1, DQA1 and DQB1 genes appear to be possible predictors of the development and recurrence of Graves' disease. Outside the HLA region, many variants of immunocompetent genes have also been identified as potential Graves' disease predictors. Apart from the immune system, some thyroid-specific genes have been described in relation to the disease. Here, we present current knowledge regarding the genetic components involved in the development and recurrence of Graves' disease. Further, we present original pilot results from a cohort of Czech Graves' disease patients regarding the HLA variants.

Parathyroid imaging by (18)F-fluorocholine PET/CT in patients with primary hyperparathyroidism and inconclusive conventional methods: clinico-pathological correlations.

(18)F-fluorocholine positron emission tomography/computed tomography (FCH) was performed after inconclusive neck ultrasound and (99)Tc-sestaMIBI SPECT (MIBI) scintigraphy in patients with primary hyperparathyroidism (PHPT) to localize abnormal parathyroid glands before surgery. The results were retrospectively evaluated and compared to postoperative histopathological findings. 13 patients with PHPT were enrolled (mean age 64.3 years, preoperative calcium 2.74 mmol/l and parathyroid hormone 114.6 ng/l). FCH localized hyperfunctioning parathyroid glands in 12 patients of 13 (per patient sensitivity 92 % and positive predictive value (PPV) 100 %). Fourteen parathyroid lesions (11 adenomas, 3 hyperplastic glands) were resected with a mean size of 11.9 mm (per lesion sensitivity 93 % and PPV 81 %). Four adenomas and one hyperplastic gland were composed of only chief cells, whereas five lesions contained both chief and oxyphil cells. In three patients an exclusively oxyphil adenoma was found, surprisingly with negative MIBI scintigraphy in spite of a high mitochondria content in the oxyphil parathyroid cells. 12 of 13 patients had thyroid disease. In our limited study sample, FCH correctly identified parathyroid adenomas and/or hyperplastic glands in 92 % of patients with previously inconclusive conventional imaging. Unlike MIBI, FCH successfully localized small, hyperplastic and multiple hyperfunctioning parathyroid glands, irrespective of their histopathological composition.

Is A163G polymorphism in the osteoprotegerin gene associated with heel velocity of sound in postmenopausal women?

Osteoprotegerin (OPG) plays an important inhibitory role in osteoclastogenesis. Polymorphisms in the OPG gene recently have been associated with various bone phenotypes including fractures. The aim of the present study was to investigate the association between three informative OPG polymorphisms and quantitative ultrasound variables of the heel. In a cohort of 165 perimenopausal women polymorphisms in the OPG promoter (A163G, T245G) and in exon 1 (G1181C) were assessed by PCR-RFLP analysis. The distribution of the investigated genotypes was similar to other Caucasian women (A163G-AA 68 %, AG 30 %, GG 2 %, T245G-TT 84.4 %, TG 15 %, GG 0.6 %, G1181C- GG 22 %, CG 55 %, CC 23 %). After adjustment for body mass index and years since menopause, in a subgroup of 87 postmenopausal subjects, calcaneal velocity of sound (VOS, m/s) was significantly associated with A163G polymorphism (p=0.0102, ANCOVA). Women with the presence of G allele (AG+GG genotypes) had significantly lower VOS than women with AA genotype. Neither T245G nor G1181C were associated with calcaneal ultrasound indices. In conclusion, A163G polymorphism was significantly associated with VOS at the heel in a limited cohort of postmenopausal women. The present study replicated in part the previous findings about OPG gene variations and peripheral bone mass in Caucasian women.

The effect of leptin on bone: an evolving concept of action.

Leptin, a cytokine-like hormone secreted by adipocytes, is known to regulate food intake but has also emerged as a significant factor in the regulation of bone mass. In humans, states of energy deprivation with low serum leptin have been associated with low bone mass. In mice, leptin deficiency led to increased trabecular bone mass with overall decrease in cortical bone. Leptin regulates bone metabolism indirectly in the hypothalamus thereby activating the sympathetic nervous system (SNS). In addition to the SNS, leptin also interacts with various hypothalamic neuropeptides, such as cocaine- and amphetamine-regulated transcript, neuropeptide Y and/or neuromedin U, which might modulate the effects of leptin on bone. In osteoblasts sympathetic signaling is further gated by the transcriptional factors called molecular clock. As a result, bone loss is accelerated showing that the central effect of leptin seems to be antiosteogenic. Additionally, leptin has a direct anabolic effect within the bone driving the differentiation of bone marrow stem cells into the osteoblastic cell lineage. Besides the interaction between the central and peripheral pathways, the overall effect of leptin on bone might be bimodal depending on leptin serum concentrations. Regulatory pathways triggering osteoblast activity might open new possibilities for anabolic treatment of osteoporosis.

Association of C/T polymorphism in the LRP5 gene with circulating follicle stimulating hormone in Caucasian postmenopausal women.

The LRP5 gene is believed to be primarily associated with bone metabolism via Wnt signaling. The latter pathway, however, appears to control various other systems outside the skeleton. To find the relationships of the LRP5 gene to serum follicle stimulating hormone (FSH) and luteinizing hormone (LH) in the cohort of normal postmenopausal women, we identified the C/T (c.4037:A1330V) polymorphism in the LRP5 gene using a restriction analysis of the PCR product in a cohort of 165 untreated pre- and post-menopausal women. In a subset of 111 post-menopausal women we analyzed the association between the LRP5 genotype and serum levels of sex-hormones including FSH and LH. The distribution of CC, TC and TT genotypes of the C/T polymorphism in the whole group was 73.9 %, 23.6 % and 2.4 %, respectively, which is comparable with other Caucasian populations. As no TT homozygote was found in the group of post-menopausal women, serum sex-hormones were compared between CC and TC genotypes. Women with the CT allele combination had markedly higher serum FSH levels as compared to carriers of the CC genotype (p<0.004). No differences between these genotypes were found in serum LH levels as well as the circulating sex-steroids such as estradiol, testosterone, dehydroepiandrosterone and/or its sulphate, androstenedione and SHBG. To conclude, the LRP5 gene is associated with circulating FSH in normal post-menopausal women in the present study. The mediating role of subtle undetectable variations in estrogen levels is discussed. We did not find any relationship between the LRP-5 genotype and serum LH levels.

Role of intron 5 C/T polymorphism of the calcium sensing receptor gene in the regulation of the serum FSH and LH in post-menopausal women.

OBJECTIVE: High extracellular calcium concentration (Cao(2+)) acts to inhibit calcium sensing receptor (CaR) signalling on cellular surfaces in parathyroid glands. This receptor is, however, also expressed on the membranes of some non-calciotropic endocrine cells, including pituitary-derived cells. The aim of our study was to analyse relationships between the CaR gene and the circulating FSH and LH in normal post-menopausal women. METHODS: A total of 95 untreated euparathyroid post-menopausal women were investigated in the study. The serum FSH and LH levels were evaluated in relationship to allele combinations of the CaR gene (C/T polymorphism in the intron 5 and A986S polymorphism in exon 7), using an analysis of co-variance (ANCOVA) model. RESULTS: Distribution of TT, TC and CC allele combinations (intron 5 C/T polymorphism) was 51, 43 and 6 %, respectively. Higher serum FSH and LH levels were found in carriers of C allele than in women without this allele (p < 0.002 and p < 0.03, respectively). No correlations were found between A986S polymorphism and serum FSH and LH levels. CONCLUSIONS: Serum FSH and LH levels are associated with intron 5 C/T (but not A986S) polymorphism of the CaR gene in untreated post-menopausal women. The physiological role of the CaR gene in the regulation of the gonadotropic function needs to be further investigated.

Postmenopausal serum androstenedione levels are associated with the calcitonin receptor gene polymorphism T1377c. A pilot study.

We tested the hypothesis that homeostasis of sex-steroids is related to the calcitonin receptor (CALCR) genotype. To determine the CALCR genotype PCR amplification followed by digestion with Alul restriction enzyme were carried out according to Nakamura et al. Indeed, a single nucleotide difference at position 1377 of cDNA generates two alleles (CC genotype or TT genotype). Serum estradiol, testosterone and their precursors androstendione (AD) and DHEA levels were estimated in a cohort of 113 postmenopausal women. While serum DHEA levels did not differ between the individual allele combinations, AD levels as well as AD/DHEA ratio were higher in carriers of TC and CC genotypes than those with TT genotype (p<0.05 and p<0.02, respectively, ANCOVA). We postulate that the 3beta-hydroxysteroid dehydrogenase activity is associated with C allele at least in C19 steroids. The data correspond with the functionality of CALCR.

Does polymorphism C1377T of the calcitonin receptor gene determine bone mineral density in postmenopausal women?

In a cross-sectional study we investigated the potential association between CALCR polymorphism (C1377T) and bone mineral density in 114 postmenopausal women. T homozygotes had higher BMD (g/cm2) at the femoral neck compared with carriers of C allele (p < 0.02, ANCOVA). Means of BMD at the lumbar spine did not differ among the genotypes (ANCOVA). In conclusion, the CALCR gene is associated with bone mass at the femoral neck in postmenopausal women.

A single nucleotide polymorphism under the reverse primer binding site may lead to BsmI mis-genotyping in the vitamin D receptor gene.

UNLABELLED: The BsmI polymorphism in the VDR gene has been extensively investigated by PCR and restriction digestion in bone genetics. A SNP within the corresponding region for the previously published reverse primer was observed and confirmed by DNA sequencing. BsmI mis-genotyping caused by this SNP could confound genetic findings. INTRODUCTION: By analyzing the FokI, BsmI, ApaI, and TaqI polymorphisms in the vitamin D receptor (VDR) gene, we observed a significantly different genotype distribution in the BsmI polymorphic locus with a deviation from Hardy-Weinberg equilibrium. One of the reasons for polymerase chain reaction (PCR) non-amplification may be a mismatched base at the primer binding region. Therefore, the aim of this study was to analyse whether a single nucleotide polymorphism (SNP), which has been recently described as TruI, is responsible for the discrepancy between expected and observed genotype frequencies. MATERIALS AND METHODS: The VDR genotypes were identified in a cohort of 165 peri- and postmenopausal women of white origin. PCR amplification was carried out using the originally published primers and followed by restriction cleavage. The BsmI genotypes were further verified with a reverse primer external to the original binding site. The presence of the TruI polymorphism under the previously published reverse primer was confirmed by a restriction digestion and DNA sequencing. In Bb subjects, the colocalization of b allele with the TruI restriction site on the same chromosome was confirmed by a simultaneous digestion of the PCR product with both BsmI and TruI restriction enzymes. RESULTS: The BsmI reanalysis with an external primer provided a higher number of heterozygous subjects with a proportionally smaller number of BB subjects, and the changed genotype distribution was under Hardy-Weinberg equilibrium (BB, 31; Bb, 80; bb, 54; r = 0.0203; p = 0.90). In our primary analysis, the presence of the TruI polymorphism led to a drop out of b allele during PCR amplification and thus to the false prevalence of BB genotypes (BB, 50; Bb, 61; bb, 54; r = 11.17; p = 0.01). CONCLUSION: The SNP in the region corresponding to the reverse primer may lead to BsmI mis-genotyping, which may have confounded some previous genetic studies.

Apolipoprotein E 4 allele is associated with low bone density in postmenopausal women.

Apolipoprotein E (Apo E) genotypes have been associated with a number of involutional disorders. Recently some studies have examined whether the Apo E 4 allele might play a role in the pathophysiology of postmenopausal osteoporosis. However, association analysis between Apo E genotypes, BMD, bone loss or fracture risk have not brought universal findings. The aim of this study was, therefore, to determine the relationship between the presence or absence of Apo E 4 allele and BMD in postmenopausal women of Caucasian origin. We studied 113 women, age 62.5 +/- 8.9 yr, who underwent measurement of hip and spine BMD by dual-energy absorptiometry (DXA, g/cm2). Apo E genotypes were assessed by PCR amplification and by restriction typing with Cfol enzyme. The Apo E allele frequencies in the study population were as follows: E2 0.084, E3 0.845, E4 0.071. Because the Apo E 4 allele was associated with osteoporosis in previous studies, the probands were dichotomized by the presence or absence of Apo E 4 allele. After adjustment for BMI and years since menopause BMD at the lumbar spine varied significantly by Apo E 4 status. Women with Apo E 4 allele had significantly lower BMD at the lumbar spine than women with no Apo E 4 allele (p<0.003, ANCOVA). In contrast, there were no significant differences in BMD at the hip comparing women with or without the Apo E 4 allele. To conclude, these data may support the importance of Apo E 4 allele in determining postmenopausal spine bone mass.

Vitamin D receptor gene polymorphisms, bone mineral density and bone turnover: FokI genotype is related to postmenopausal bone mass.

The relationship between vitamin D receptor (VDR) intragenic polymorphisms FokI, BsmI, ApaI and TaqI and bone mineral density (BMD) or biochemical markers of bone remodeling were investigated in 114 Czech postmenopausal women, on the average 62.5+/-8.9 years of age. Restriction fragment length polymorphisms in the VDR gene were assessed by PCR amplification and digestion with restriction enzymes FokI, BsmI, ApaI, and TaqI recognizing polymorphic sites in the VDR locus. Bone mineral density was measured at the lumbar spine and at the hip by dual-energy X-ray absorptiometry (DEXA, g/cm2). After adjusting for age and the body mass index (BMI), subjects with the ff genotype had 9.4% lower BMD at the hip than those with the Ff genotype (p=0.0459, Tukey's test). FF individuals had an intermediate BMD at the hip. A similar pattern of lower lumbar spine BMD was also found in ff individuals, but it did not reach statistical significance. There was no relationship between BsmI, ApaI and TaqI VDR polymorphisms and BMD at any skeletal site. Subjects with Aa (ApaI) genotypes had higher levels of propeptide of type I collagen (PICP) than homozygous AA (p=0.0459, Tukey's test). In FokI, BsmI and TaqI restriction sites the biochemical markers of bone remodeling did not differ by genotype. In addition, no significant difference was observed in VDR genotypic distribution between osteoporotic women and non-osteoporotic controls in the study group. To conclude, the FokI genotype of the vitamin D receptor gene is related to bone mass at the hip in Czech postmenopausal women, whereas the importance of remaining VDR genotypes was not evident.

The estrogen receptor alpha gene determines serum androstenedione levels in postmenopausal women.

Estrogen receptors (ER) are expressed not only in the reproductive system and ovaries but also in some other tissues, including the adrenal gland. The purpose of this study was to assess the association between the estrogen receptor (ER) alpha gene polymorphisms XbaI and PvuII and circulating levels of androstenedione, a precursor of sex-steroids synthetized in the ovary and adrenal. After adjustment for years since menopause, body mass, and dehydroepiandrosterone (DHEA) levels, a highly significant relationship was demonstrated between androstenedione and XbaI or PvuII polymorphisms, the highest levels of the hormone being found in the xx and pp genotypes (P<0.05 as compared to XX or PP, ANCOVA followed by least significant difference (LSD) multiple comparisons). This suggests that the ER genotype may determine the function of the sex-steroid system not only at the receptor level but also at the level of hormone synthesis. The pathogenetic role of this association in diseases related to menopause, such as osteoporosis, remains to be determined.

Dehydroepiandrosterone status in postmenopausal women is determined by the gene for the vitamin D receptor.

Data documenting the indirect interaction of vitamin D and bone metabolism via hormonal systems are rare. The authors analysed the predictive role of the vitamin D receptor (VDR) gene for circulating sex steroids and their precursors in postmenopausal women. Using the PCR technique, the polymorphic FokI, ApaI, TaqI and BsmI sites of the VDR gene were determined in relation to serum dehydroepiandrosterone sulphate (DHEAS), androstenedione (AD), testosterone, and estradiol levels. After adjustment to body mass and years since menopause, circulating DHEAS was higher in the Ff genotype than in ff (p < 0.001) and FF genotypes (p < 0.05, ANCOVA followed by least significant difference multiple comparison tests). The Ff genotype also contributed to the highest BMD at the hip (p < 0.01 as compared to ff genotype) and at the spine (p < 0.05). No significant associations were found between ApaI, TaqI and BsmI polymorphisms and serum DHEAS or between FokI, ApaI, TaqI or BsmI and serum androstenedione, testosterone or estradiol. The study shows that the VDR gene predicts synthesis and/or metabolism of sexual steroid preursor DHEA in parallel with bone mineral density (BMD). The results indicate that DHEA production and bone mass share a common genetic control through VDR.