The Y606C RET mutation causes a receptor gain of function.

CONTEXT: Medullary thyroid carcinoma (MTC) is the most common feature of multiple endocrine neoplasia type 2A (MEN2A) and occurs in almost all patients affected by germline RET mutations. OBJECTIVE: We identified and characterized an activating germline RET point mutation (G>A substitution leading to the heterozygous missense mutation Y606C in exon 10), in a 58-year-old female affected by MTC. DESIGN: The RET/Y606C and RET/C620Y, obtained by site-directed mutagenesis, as well as the RET/wild-type (wt) were cloned in an expression vector and transiently transfected in NIH3T3 fibroblasts. In vitro cell model was used to evaluate the effect of Y606C mutation on the RET downstream signalling pathways through Western blot analysis. RESULTS: We found that the cysteine insertion, due to the Y606C mutation, results in increased receptor dimerization, which is accompanied by an increased tyrosine phosphorylation of the Y905 residue in the RET/Y606C, demonstrating that the Y606C mutation is associated with constitutive receptor activation. As RET activation results in an intracellular signalling cascade involving extracellular signal-regulated kinases (ERKs), we investigated ERK activity in our transfected cells. Results demonstrated a significant increase in ERK2 phosphorylation in the RET/Y606C vs. the RET/wt and RET/C620Y transfected cells, suggesting an up-regulation of RET signalling. CONCLUSIONS: All these findings demonstrate that the Y606C mutation is associated with RET constitutive activation and thus has to be considered of pathogenetic relevance in the development of MTC.

Genetics and biology of pheochromocytoma.

The familial forms of pheochromocytoma have recently been demonstrated to be more frequent than believed in the past. The genes currently known to be responsible for tumor formation are RET, VHL, NF1, SDHB, SDHC and SDHD. Germline mutations of these genes increase the risk of developing pheochromocytomas and/or paragangliomas which variably associate with other neoplasms and characterize diverse clinical syndromes such as MEN 2, von Hippel-Lindau (VHL), and neurofibromatosis type 1 (NF 1), or the PGL syndromes, respectively. Although the pathogenesis of pheochromocytoma/paraganglioma formation is still largely unknown, studies of the familial forms have started to uncover some pathways that favor tumor formation, such as activation of tyrosine-kinase, induction of hypoxia-inducible factors, activation of the oncogene Ras or reduced apoptosis. These studies have also demonstrated that various gene mutations can differently affect the biological characteristics of pheochromocytoma: for example, while the tumors are mostly adrenergic (epinephrine secreting) and episodically secreting in MEN 2, they are mostly noradrenergic (norepinephrine secreting) and continuously secreting in VHL. Biological variability can also be observed in the PGL syndromes where tumors develop in the head and neck and are parasympathetic in origin and non-secreting, or in the thorax and the abdomen, where they are sympathetic in origin and catecholamine secreting. Genetic testing in patients with pheochromocytomas or paragangliomas is, at present, strongly recommended and is mandatory in young patients or in cases of multiple or recurrent tumors. The clinical picture and the biological characteristics of the tumor may suggest the priority of the genes to be tested first.

SDH mutations in patients affected by paraganglioma syndromes: a personal experience.

Mutations in genes encoding mitochondrial succinate dehydrogenase (SDH) are frequently involved in the development of neural crest-derived (NCD) tumors, such as pheochromocytomas (PHEOs) or paragangliomas (PGLs). In this study we report the results of sequencing analysis in leukocyte DNA of patients affected by PHEO/PGL who turned out to be SDH mutation carriers. A nonsense germline heterozygous mutation (Q109X) was found in the exon 4 of the SDHD gene in the index cases of six unrelated families affected by PHEO/PGL. Haplotype analysis showed the presence of a founder effect. Affected patients showed high clinical variability, ranging from monolateral to bilateral glomus tumors, variably associated or not with PGLs or PHEOs. A novel missense SDHD variant, T112I, was also found in one of our families. A new missense G106D mutation, involving a highly conserved amino acid, was found in two sisters affected by bilateral glomus tumors. A P81L mutation associated with abdominal and head and neck PGL was detected in three families. A G12S variant of the SDHD gene was found in one patient affected by a PHEO. The finding of this variant in 3 of 100 control subjects suggests that it is a polymorphism and not a mutation. A novel IVS2-1G>T variant was found at intron 2 of SDHD gene in one patient affected by a glomus tumor. All the tumors associated with SDHD mutations were benign. Conversely, the only mutation we found in SDHB gene (IVS3+1G>A) was associated with a malignant PHEO.

Estrogen receptor gene polymorphisms and the genetics of osteoporosis: a HuGE review.

Osteoporosis (OMIM166710) is a common skeletal disorder characterized by low bone mass and microarchitectural deterioration of bone tissue with increased susceptibility to fracture. Osteoporosis has a complex etiology and is considered a multifactorial polygenic disease in which genetic determinants are modulated by hormonal, environmental, and nutritional factors. Estrogens are known to play an important role in regulating bone homeostasis and preventing postmenopausal bone loss. They act through binding to two different estrogen receptors (ERs), ER alpha (OMIM133430) and ER beta (OMIM601663), which are members of the nuclear receptor superfamily of ligand-activated transcription factors. Different polymorphisms have been described in both the ER alpha and ER beta genes. Although a large number of association studies have been performed, the individual contribution of these polymorphisms to the pathogenesis of osteoporosis remains to be universally confirmed. Moreover, an important aim in future work will be to define their functional molecular consequences and their interaction with the environment in the causation of the osteoporotic phenotype. A further promising application of these polymorphisms comes from their pharmacogenomic implications, with the possibility of providing better guidance for therapeutic regimens, such as estrogen replacement therapy and selective ER modulators. At the moment, no recommendations for population-based screening can be made.

DAZL polymorphisms and susceptibility to spermatogenic failure: an example of remarkable ethnic differences.

Polymorphisms in genes involved in spermatogenesis are considered potential risk factors for male infertility. Recently a polymorphism in the deleted in azoospermia-like (DAZL) gene (T54A) was reported as susceptibility factor to oligo/azoospermia in the Chinese population. DAZL is an autosomal homologue of the Y chromosomal DAZ (deleted in azoospermia) gene cluster and both are considered master regulators of spermatogenesis. The aim of the present study was to screen (i) for mutations of the entire coding sequence of the DAZL gene in patients lacking of the DAZ gene cluster, in order to evaluate if DAZL polymorphisms may influence the AZFc deletion phenotype; (ii) for the two previously described (and eventually newly identified) single nucleotide polymorphisms (SNPs) in a large group of infertile and normospermic men of Italian origin. We failed to detect new mutations. We confirmed previous results showing no evidence for a functional role of the T12A mutation. Surprisingly, the T54A polymorphism, which was present in 7.4% of the Chinese patients was absent in our Caucasian population. This remarkable difference represent an example of how ethnic background is important also for polymorphisms involved in spermatogenesis and contributes to better select clinically relevant tests, specifically based on the ethnic origin of the infertile patients.

The clinical significance of the POLG gene polymorphism in male infertility.

Based on association studies, an increasing number of gene polymorphisms have been proposed as modulators of spermatogenesis. Interestingly, a clear cause-effect relationship between a polymorphism of the POLG gene and oligo(astheno)zoospermia was recently described. The POLG gene contains a polymorphic CAG repeat, and the presence of a homozygous mutant (not10/not10 CAG) genotype was found only in infertile men. In the present study, a large number of infertile patients and normospermic men of Italian origin were studied to define the effect of POLG genotypes on spermatogenic potential and whether the homozygous mutant is specific for spermatogenic disturbances. The mutated genotype was found at the same frequency in both infertile and normospermic men. Mean values of sperm parameters such as sperm count, motility, and morphology did not differ significantly between carriers of the three different genotypes. Our study failed to confirm any influence of the POLG gene polymorphism on the efficiency of the spermatogenesis. More importantly, considering that the homozygous mutant genotype has been found in normospermic fertile men, the analysis of the CAG repeat tract of the POLG gene does not appear to have any clinical diagnostic value.

Genetics of osteoporosis: role of steroid hormone receptor gene polymorphisms.

Osteoporosis is a common skeletal disease characterized by low bone mass and microarchitectural deterioration of bone tissue with a consequent increase in bone fragility and susceptibility to fracture. In the past years, twin and family study have shown that this disease recognizes a strong genetic component and that genetic factors play an important role in regulating bone mineral density (BMD). While in few isolate conditions osteoporosis can be inherited in a simple Mendelian pattern, due to single gene mutations, in the majority of cases has to be considered a multifactorial polygenic disease in which genetic determinants are modulated by hormonal, environmental and nutritional factors. Given the important role that steroid hormones play in bone cell development and in the maintenance of normal bone architecture, polymorphisms at receptor of the steroid/thyroid hormone receptor superfamily, such as estrogen receptor alpha (ERalpha) and Vitamin D receptor (VDR) have been thoroughly investigated in the last years and appeared to represent important candidate genes. The individual contribution of these genetic polymorphisms to the pathogenesis of osteoporosis remains to be universally confirmed and an important aim in future work will be to define their functional molecular consequences and how these polymorphisms interact with each other and with the environment to cause the osteoporotic phenotype. A further promising application of genetic studies in osteoporosis comes from their pharmacogenomic implications, with the possibility to give a better guidance for therapeutic agents commonly used to treat this invalidating disorder or to identify target molecules for new therapeutic agents.

Genetic markers of osteoarticular disorders: facts and hopes.

Osteoarthritis and osteoporosis are the two most common age-related chronic disorders of articular joints and skeleton, representing a major public health problem in most developed countries. Apart from being influenced by environmental factors, both disorders have a strong genetic component, and there is now considerable evidence from large population studies that these two disorders are inversely related. Thus, an accurate analysis of the genetic component of one of these two multifactorial diseases may provide data of interest for the other. However, the existence of confounding factors must always be borne in mind in interpreting the genetic analysis. In addition, each patient must be given an accurate clinical evaluation, including family history, history of drug treatments, lifestyle, and environment, in order to reduce the background bias. Here, we review the impact of recent work in molecular genetics suggesting that powerful molecular biology techniques will soon make possible both a rapid accumulation of data on the genetics of both disorders and the development of novel diagnostic, prognostic, and therapeutic approaches.

Polymorphism of the aromatase gene in postmenopausal Italian women: distribution and correlation with bone mass and fracture risk.

Conversion of C(19) steroids to estrogens is catalyzed by the aromatase enzyme. Inactivating mutations of the aromatase gene are associated with decreased bone mineral density in both men and women. Genetic studies suggest that several genes contribute to the regulation of bone mass via interaction with the modeling and remodeling processes. Among these genes, the aromatase gene is a potential candidate to be evaluated for segregation with bone metabolism and bone mass. A tetranucleotide simple tandem repeat polymorphism in intron 4 at the human aromatase cytochrome P-450 gene has been recently described. In the present study we evaluated the distribution of this polymorphism in a cohort of Italian postmenopausal women, both normal and osteoporotic. We observed that the NN genotype was significantly more frequent in nonosteoporotic women than in osteoporotic women (72.7% vs. 27.2%), whereas the DN genotype was significantly more represented in osteoporotic women (90.48% vs. 9.5%; Pearson's chi(2) test = 42.8; df = 10; P = or < 0.01). The allele containing the longer TTTA repeats was statistically more represented in nonosteoporotic women (Pearson's chi(2) test = 19.14; df = 2; P = 0.00007). In addition, women with a high number of TTTA repeats had a significantly higher lumbar bone mineral density than women with alleles containing 8-11 TTTA repeats (P = 0.03). Finally, considering the spine fractures, a significantly higher incidence was observed in women with shorter TTTA repeats than in those with longer TTTA repeats (Pearson's chi(2) test = 7.3; df = 2; P = 0.02), equivalent to a relative risk of 4.1 (95% confidence interval, 1.19-13.87). In conclusion, the aromatase gene can be one of the several genes potentially involved in the maintenance of bone mass and in the regulation of bone mass loss.

Genotype distribution of estrogen receptor-alpha gene polymorphisms in Italian women with surgical uterine leiomyomas.

OBJECTIVE: To explore a possible association between estrogen receptor-alpha (ER-alpha) gene polymorphisms and development of uterine leiomyomas. DESIGN: Case-control study. SETTING: University teaching hospital. PATIENT(S): 119 women with clinically and surgically diagnosed uterine leiomyomas. INTERVENTION(S): Therapeutic hysterectomy. MAIN OUTCOME MEASURE(S): Frequency and distribution of ER-alpha gene polymorphisms. RESULT(S): No statistically significant differences between controls and patients in the allele frequency and genotype distribution were found when Pvu II and Xba I restriction polymorphism sites were analyzed separately. When the two ER-alpha gene polymorphisms were analyzed in combination, five major genotypes were recognized in controls or patients; the frequency differed slightly but not significantly between groups. CONCLUSION(S): The Pvu II and Xba I polymorphisms in the ER-alpha gene do not produce different risks of developing uterine leiomyomas.

Evidence of a linkage disequilibrium between polymorphisms in the human estrogen receptor alpha gene and their relationship to bone mass variation in postmenopausal Italian women.

Bone mineral density (BMD), the major determinant of osteoporotic fracture risk, has a strong genetic component. The discovery that inactivation of estrogen receptor alpha (ERalpha) gene is associated with low BMD indicated ERalpha as a candidate gene for osteoporosis. We have investigated the role of three ERalpha gene polymorphisms [intron 1 PVU:II and XBA:I RFLPs and TA dinucleotide repeat polymorphism 5' upstream of exon 1] in 610 postmenopausal women. There was a strong linkage disequilibrium between intron 1 polymorphic sites and also between these sites and the microsatellite (TA)(n) dinucleotide polymorphism, with a high degree of coincidence of the short TA alleles and the presence of PVU:II and XBA:I restriction sites. No significant relationship between intron 1 RFLPs and BMD was observed. A statistically significant correlation between (TA)(n) repeat allelic variants and lumbar BMD was observed (P = 0.04, ANCOVA), with subjects with a low number of repeats (TA < 15) showing the lowest BMD values. We observed a statistically significant difference in the mean +/- SD number of TA repeats between analyzed women with a vertebral fracture (n = 73) and the non-fracture group, equivalent to 2.9 (95% CI 1.56-5.72) increased fracture risk in women with a low number of repeats (TA < 15). We conclude that in this large population sample the (TA)(n) dinucleotide repeat polymorphism at the 5' end of the ERalpha gene accounts for part of the heritable component of BMD and might prove useful in the prediction of vertebral fracture risk in postmenopausal osteoporosis.

Absence of correlation between BMP-4 polymorphism and postmenopausal osteoporosis in Italian women.

Several studies have demonstrated that bone has the power of regeneration and repair. BMPs (bone morphogenetic proteins) are involved in the determination of osteoblast phenotype and bone turnover, therefore genes coding for these proteins, like BMP-4, could be considered potential candidate genes for osteoporosis. We investigated the association of BMP-4 gene polymorphism with osteoporosis in a cohort of 72 osteoporotic, postmenopausal women and 82 unrelated controls. We failed to detect any significant association between this genetic marker and the disease.

The biphasic effects of nitric oxide in primary rat osteoblasts are cGMP dependent.

Nitric oxide is a gas radical regulating cell behaviour in the cardiovascular, immune, and central nervous systems. It has now been established as an important signalling molecule in bone. However, the effects of this gas radical on osteoblastic function are still unclear; in fact, while NO seems to be involved in anabolic processes mediated by mechanical strain, sex hormones and fracture healing, it also mediates catabolic processes in response to inflammation. We show here that a slow and moderate release of nitric oxide stimulates the replication of primary rat osteoblasts and alkaline phosphatase activity, while a rapid release and high concentrations of NO inhibit proliferation and induce apoptosis. We demonstrate that both the stimulatory and apoptosis-inducing effects of NO on primary osteoblasts are mediated by the second messenger cGMP, since both are abolished by the guanylate cyclase inhibitor ODQ.

MEN1 gene mutation analysis in Italian patients with multiple endocrine neoplasia type 1.

Multiple endocrine neoplasia type 1 (MEN 1) is a familial syndrome characterized by parathyroid, enteropancreatic and pituitary tumors. The gene responsible for this syndrome is localized at chromosomal 11q13 region and DNA markers from this region cosegregate with the disease. The recent identification of the MEN1 gene, encoding for a protein termed menin of 610 amino acids, allowed mutational screening to be performed both in affected families and sporadic cases. To date many different heterozygous mutations, spreading across all the encoding sequence, have been identified in MEN 1 patients with no apparent mutational hot spots or genotype-phenotype correlation. To analyze the genetic alterations of the MEN1 gene occurring in Italian patients we performed mutational screening by Denaturant Gradient Gel Electrophoresis followed by sequencing of exons 2-10 of the MEN1 gene in 27 Italian MEN 1 families and in five sporadic cases. We identified 17 different heterozygous mutations in 60% of analyzed cases. Twelve of these mutations are novel. Two mutations each occurred twice in unrelated families but no evidence of genotype-phenotype correlation can be established for these families. The extension of genetic diagnosis to asymptomatic family members allowed the identification of 10 MEN1 mutant gene carriers, one newly described and nine previously detected by linkage analysis with DNA markers from the 11q13 region. Our findings add new information to the diversity of mutations occurring in the MEN1 gene and confirm that the mutational screening of MEN 1 is a useful approach to detect individuals at higher risk of developing MEN 1-associated tumors.

Telomerase activity is significantly enhanced in malignant adrenocortical tumors in comparison to benign adrenocortical adenomas.

Telomerase is an enzyme that causes short repeated sequence addition to the ends of chromosomes, thereby preventing their shortening during cell division and counteracting cell senescence. Telomerase activity is generally absent in adult differentiated cells, whereas it has been demonstrated in tumor cells, suggesting that its presence might be considered an index of malignancy. To evaluate whether telomerase might be considered a good predictive index of malignancy in adrenocortical tumors, we measured telomerase activity in 11 adrenal adenomas and 7 carcinomas obtained at surgery, using an original quantitative method. Telomerase activity was significantly higher (P<0.001) in carcinomas than in adenomas (median, 15.2 ng DNA/microg protein; range, 9.0-27.6 vs. 2.0; range, 0-8.3), and no overlap was observed between the 2 groups. In carcinomas, telomerase activity was significantly correlated with tumor diameter (r = 0.939; P<0.0001), whereas in adenomas it was not. The results of this study suggest that quantitative telomerase measurement may represent a useful tool to differentiate malignant from benign adrenocortical tumors.

Association of the estrogen receptor alpha gene polymorphisms with sporadic Alzheimer's disease.

Alzheimer's disease (AD) is a multifactorial disorder determined by the interaction of genetic, metabolic, and environmental factors. In the common late-onset familial and sporadic forms of AD apolipoprotein E type 4 allele (APOE-epsilon4) is now widely accepted as a major risk factor. The association of estrogen treatment with a reduction in the risk of AD together with the modulation by estrogen of the secretory metabolism of the amyloid precursor protein offers new possibilities for identification of other AD susceptibility genes, as those encoding for the estrogen receptors (ERs). A total of 193 patients with sporadic late-onset AD, meeting the NINCDS-ADRDA criteria, and a total of 202 control subjects, age and education matched, were included in this study. PvuII and XbaI ERalpha and HhaI APOE gene polymorphisms were evaluated in genomic DNA by Polymerase Chain Reaction (PCR). The frequency of the various ERalpha genotypes by the combination of P, p and X, x was calculated for controls and AD patients stratified based on ApoE typing. When the two ERalpha gene polymorphisms were analyzed in combination, 7 genotypes were recognized, with a significantly increased prevalence of PPXX genotype in AD patients compared to controls (P = 0.0001). Risk of AD increased by a factor of 7.6 (CI [1.10-62.3]) in homozygous APOE-epsilon4 individuals with PPXX ERalpha genotype. These results are consistent with a segregation of PPXX ERalpha genotype with a higher risk of developing late-onset sporadic AD in the Italian population. The ERalpha gene appears to interact with the APOE-epsilon4 genotype in determining AD susceptibility.

Telomerase repeat amplification protocol (TRAP): A new molecular marker for parathyroid carcinoma.

Telomerase results to be active in human germ, stem cells, several malignant cell tumors and in immortalized cell lines. In order to investigate if molecular mechanisms other than Rb gene inactivation can be helpful to diagnose malignancy of parathyroid tumors, we decided to investigate the presence of active telomerase in homogenates from different pathological parathyroid tissues (hyperplastic, adenomatous, carcinomatous, and normal) and primary cell cultures. The TRAP assay was performed to detect this activity in histologically characterized normal, hyperplastic, adenomatous, and carcinomatous human parathyroid tissues, primary cell lines, and one metastatic tissue from parathyroid carcinoma. Only malignant parathyroid glands and the metastatic tissue were TRAP positive. Our findings suggest that telomerase expression could represent an important molecular mechanism underlying the acquisition and progression of an aggressive phenotype of epithelial parathyroid cells and it may help to predict their malignant potential. The TRAP assay is easy to perform and it could become an additional tool to be included in the harmamentarium for the molecular diagnosis of parathyroid carcinoma.

FokI polymorphism at translation initiation site of the vitamin D receptor gene predicts bone mineral density and vertebral fractures in postmenopausal Italian women.

A novel T/C polymorphism (ATG to ACG) at the translation initiation site of the vitamin D receptor (VDR) gene, defined by FokI restriction endonuclease, has been recently associated with variation in bone mineral density (BMD) and rates of bone loss in a group of postmenopausal Mexican-American women. The presence of the restriction site, designated as f, allows protein translation to initiate from the first ATG, while the allele lacking the site, indicated as F, initiates translation at a second ATG. In this study, we investigated the role of FokI polymorphism in a group of 400 postmenopausal women of Italian descent stratified for BMD into osteoporotic (n = 164), osteopenic (n = 117), and normal (n = 119) groups. There were 159 (41%) FF homozygotes, 55 (14%) ff homozygotes, and 186 (45%) Ff heterozygotes. In the whole population, we observed a weak association between FokI polymorphism and lumbar BMD (p = 0.06, analysis of covariance [ANCOVA]) but not with femoral neck BMD (p = 0.5, ANCOVA). Interestingly, the effect of FokI genotypes on lumbar BMD was influenced by the years since menopause such that differences in BMD related to different VDR allelic variants were greater among women in the first 5 years of menopause (p = 0.04, ANCOVA), progressively declining afterward. In addition, a significantly higher prevalence of ff genotype in osteoporotic than in osteopenic and normal women was observed (p = 0.04, Chi-square test). Finally, ff genotype resulted significantly over-represented in the group of women with a vertebral fracture as compared with controls (p = 0.003, Chi-square test), equivalent to a relative risk of 2.58 (95% confidence intervals 1.36-4.91). We conclude that in this population, FokI polymorphism at the VDR gene locus accounts for a part of the heritable component of BMD at the lumbar spine.

Rapid, quantitative nonisotopic assay for telomerase activity in human tumors.

Telomerase is a ribonucleoprotein enzyme that adds TTAGGG repeats onto human telomeres, preventing their shortening. The activation of this enzyme is an important step in cell immortalization and carcinogenesis and seems to represent a new and promising marker in cancer diagnosis and management. Telomerase activity is usually detected in cellular protein extract by the telomeric repeat amplification protocol (TRAP) assay, which can provide only a qualitative (presence/absence) evaluation. Here we present a modification of this method that can provide quantitative information without requiring time-consuming post-PCR procedures such as gel electrophoresis with radioactive materials and autoradiography. The detection and measurement of telomerase activity is performed by evaluating the amount of double-stranded DNA generated in the telomerase reaction and PCR amplification, with the use of the sensitive DNA fluorescent dye PicoGreen. In a subset of tumors, the presence of telomerase activity was confirmed by the conventional TRAP assay. By this method we evaluated telomerase activity in unselected groups of breast (n = 15), ovarian (n = 12), endometrial (n = 12), gastric (n = 20), and renal (n = 12) carcinomas, in meningiomas (n = 8), and in pheochromocitomas (n = 10). The results indicate substantial differences of telomerase activity among cancer groups; however, a large variability among patients of the same group is observed. Kidney, ovarian, and breast carcinomas showed the highest mean values (31.8 +/- 28.9, 29.2 +/- 26.7, and 35.3 +/- 15.9 ng DNA/microg protein, respectively, mean +/- SD), whereas gastric and endometrial cancers had a lower activity (17.2 +/- 8.8 and 13.5 +/- 7.9 ng DNA/microg protein, respectively). Very low or no detectable telomerase activity was found in meningiomas (with the exception of one malignant atypical variant) and pheochromocitomas (9.7 +/- 12.9 and 2.8 +/- 2.1 ng DNA/microg protein, respectively). In conclusion, our method seems to be an accurate and reasonable procedure for measuring telomerase activity in human cancers.