Hip fracture trends in the United States, 2002 to 2015.

An analysis of United States (US) Medicare claims data from 2002 to 2015 for women aged ≥ 65 years found that age-adjusted hip fracture rates for 2013, 2014, and 2015 were higher than projected, resulting in an estimated increase of more than 11,000 hip fractures. INTRODUCTION: Hip fractures are a major public health concern due to high morbidity, mortality, and healthcare expenses. Previous studies have reported a decrease in the annual incidence of hip fractures in the US beginning in 1995, coincident with the introduction of modern diagnostic tools and therapeutic agents for osteoporosis. In recent years, there has been less bone density testing and fewer prescriptions for osteoporosis treatments. The large osteoporosis treatment gap raises concern of possible adverse effects on hip fracture rates. METHODS: We assessed hip fracture incidence in the US to determine if the previous decline in hip fracture incidence continued. Using 2002 to 2015 Medicare Part A and Part B claims for women ≥ 65 years old, we calculated age-adjusted hip fracture rates, weighting to the 2014 population. RESULTS: We found that hip fracture rates declined each year from 2002 to 2012 and then plateaued at levels higher than projected for years 2013, 2014, and 2015. CONCLUSIONS: The plateau in age-adjusted hip fracture incidence rate resulted in more than 11,000 additional estimated hip fractures over the time periods 2013, 2014, and 2015. We recommend further study to assess all factors contributing to this remarkable change in hip fracture rate and to develop strategies to reduce the osteoporosis treatment gap.

Correction to: Hip fracture trends in the United States, 2002 to 2015.

The name of the first author, E.M. Lewiecki, was rendered incorrectly in the original publication. The publisher regrets any inconvenience and is pleased to correct the error here.

Inhibition of growth hormone receptor activation by pegvisomant may increase bone density in acromegaly.

Treatment of acromegaly with pegvisomant lowers serum IGF-1 and raises serum growth hormone. As both IGF-1 and GH are important for bone growth and remodeling, we were concerned that lowering of IGF-1 could cause loss of bone. To evaluate the effects of treatment of acromegaly with pegvisomant on bone mineral density (BMD) we developed an observational, prospective study. 7 acromegaly patients participated in the study. Male and female subjects aged 18 years or more were eligible to participate. Patients were eugonadal or on adequate gonadal replacement therapy for at least 3 years before participating in the study. These patients were treated with a mean dosage of 20 mg of pegvisomant daily for up to 7 years. Bone mineral density (BMD) was evaluated by dual X-ray absorbtiometry (DXA) at baseline, 8, and 18 months as a part of a prospective trial and periodically thereafter. Baseline mean serum insulin-like growth factor-1 (IGF-1) concentration±SD was elevated in all patients (679.86±138.21 ng/ml). The IGF-1 concentrations at 18 months decreased significantly from baseline (p=0.016). Wilcoxon signed-rank tests showed significant increases in the spine BMD from baseline to 18 months (p=0.016) and significant increases in the right hip BMD from baseline to 18 months (p=0.032). The range of the increases was 4.3-17.8% at 7 years. It is concluded that successful treatment of acromegaly with pegvisomant increases BMD.

Beyond RET: potential therapeutic approaches for advanced and metastatic medullary thyroid carcinoma.

Medullary thyroid carcinoma (MTC) is a rare calcitonin-producing neuroendocrine tumour that originates from the parafollicular C-cells of the thyroid gland. The RET proto-oncogene encodes the RET receptor tyrosine kinase, which has essential roles in cell survival, differentiation and proliferation. Activating mutations of RET are associated with the pathogenesis of MTC and have been demonstrated in nearly all hereditary and in 30-50% of sporadic MTC cases, making this receptor an excellent target for small-molecule inhibitors for this tumour. Clinical trials of small organic inhibitors of tyrosine kinase receptors (TKIs) targeting the RET receptor have shown efficacy for treatment of metastatic MTC with 30-50% of patients responding to these agents. Despite the importance of the RET receptor in MTC, it is clear that other signal transduction pathways, tyrosine kinase receptors, and tumour suppressor genes are involved in MTC tumourigenesis and progression. A better understanding of molecular cross-talk between these signal pathways and the RET receptor may lead to combinatorial therapy that will improve outcomes beyond what is currently possible with RET-directed TKIs. Finally, there is evidence that immunological-based therapy using dendritic cell vaccination strategies have been effective for reducing tumour mass in a small number of patients. The identification of additional MTC-specific tumour antigens and a better understanding of specific epitopes in these tumour antigens may lead to improvement of response rates.

Mutations in the sulfonylurea receptor gene in familial persistent hyperinsulinemic hypoglycemia of infancy.

Familial persistent hyperinsulinemic hypoglycemia of infancy (PHHI), an autosomal recessive disorder characterized by unregulated insulin secretion, is linked to chromosome 11p14-15.1. The newly cloned high-affinity sulfonylurea receptor (SUR) gene, a regulator of insulin secretion, was mapped to 11p15.1 by means of fluorescence in situ hybridization. Two separate SUR gene splice site mutations, which segregated with disease phenotype, were identified in affected individuals from nine different families. Both mutations resulted in aberrant processing of the RNA sequence and disruption of the putative second nucleotide binding domain of the SUR protein. Abnormal insulin secretion in PHHI appears to be caused by mutations in the SUR gene.

Alternative ribonucleic acid processing in endocrine systems.

Alternative RNA processing is a mechanism for creation of protein diversity through selective inclusion or exclusion of RNA sequence during posttranscriptional processing. More than one-third of human pre-mRNAs undergo alternative RNA processing modification, making this a ubiquitous biological process. The protein isoforms produced have distinct and sometimes opposite functions, underscoring the importance of this process. This review focuses on important endocrine genes regulated by alternative RNA processing. We discuss how diverse events such as spermatogenesis or GH action are regulated by this process. We focus on several endocrine (calcitonin/calcitonin gene-related peptide) and nonendocrine (Drosophila doublesex and P-element and mouse c-src) examples to highlight recent progress in the elucidation of molecular mechanisms regulating this process. Finally, we outline methods (model systems and techniques) used by investigators in this field to study processing of individual pre-mRNAS:

An intron enhancer containing a 5' splice site sequence in the human calcitonin/calcitonin gene-related peptide gene.

Regulation of calcitonin (CT)/calcitonin gene-related peptide (CGRP) RNA processing involves the use of alternative 3' terminal exons. In most tissues and cell lines, the CT terminal exon is recognized. In an attempt to define regulatory sequences involved in the utilization of the CT-specific terminal exon, we performed deletion and mutation analyses of a mini-gene construct that contains the CT terminal exon and mimics the CT processing choice in vivo. These studies identified a 127-nucleotide intron enhancer located approximately 150 nucleotides downstream of the CT exon poly(A) cleavage site that is required for recognition of the exon. The enhancer contains an essential and conserved 5' splice site sequence. Mutation of the splice site resulted in diminished utilization of the CT-specific terminal exon and increased skipping of the CT exon in both the mini-gene and in the natural CT/CGRP gene. Other components of the intron enhancer modified utilization of the CT-specific terminal exon and were necessary to prevent utilization of the 5' splice site within the intron enhancer as an actual splice site directing cryptic splicing. Conservation of the intron enhancer in three mammalian species suggests an important role for this intron element in the regulation of CT/CGRP processing and an expanded role for intronic 5' splice site sequences in the regulation of RNA processing.

Polypyrimidine tract-binding protein positively regulates inclusion of an alternative 3'-terminal exon.

Polypyrimidine tract-binding protein (PTB) is an abundant vertebrate hnRNP protein. PTB binding sites have been found within introns both upstream and downstream of alternative exons in a number of genes that are negatively controlled by the binding of PTB. We have previously reported that PTB binds to a pyrimidine tract within an RNA processing enhancer located adjacent to an alternative 3'-terminal exon within the gene coding for calcitonin and calcitonin gene-related peptide. The enhancer consists of a pyrimidine tract and CAG directly abutting on a 5' splice site sequence to form a pseudoexon. Here we show that the binding of PTB to the enhancer pyrimidine tract is functional in that exon inclusion increases when in vivo levels of PTB increase. This is the first example of positive regulation of exon inclusion by PTB. The binding of PTB was antagonistic to the binding of U2AF to the enhancer-located pyrimidine tract. Altering the enhancer pyrimidine tract to a consensus sequence for the binding of U2AF eliminated enhancement of exon inclusion in vivo and exon polyadenylation in vitro. An additional PTB binding site was identified close to the AAUAAA hexanucleotide sequence of the exon 4 poly(A) site. These observations suggest a dual role for PTB in facilitating recognition of exon 4: binding to the enhancer pyrimidine tract to interrupt productive recognition of the enhancer pseudoexon by splicing factors and interacting with the poly(A) site to positively affect polyadenylation.

Regulation of alternative polyadenylation by U1 snRNPs and SRp20.

Although considerable information is currently available about the factors involved in constitutive vertebrate polyadenylation, the factors and mechanisms involved in facilitating communication between polyadenylation and splicing are largely unknown. Even less is known about the regulation of polyadenylation in genes in which 3'-terminal exons are alternatively recognized. Here we demonstrate that an SR protein, SRp20, affects recognition of an alternative 3'-terminal exon via an effect on the efficiency of binding of a polyadenylation factor to an alternative polyadenylation site. The gene under study codes for the peptides calcitonin and calcitonin gene-related peptide. Its pre-mRNA is alternatively processed by the tissue-specific inclusion or exclusion of an embedded 3'-terminal exon, exon 4, via factors binding to an intronic enhancer element that contains both 3' and 5' splice site consensus sequence elements. In cell types that preferentially exclude exon 4, addition of wild-type SRp20 enhances exon 4 inclusion via recognition of the intronic enhancer. In contrast, in cell types that preferentially include exon 4, addition of a mutant form of SRp20 containing the RNA-binding domain but missing the SR domain inhibits exon 4 inclusion. Inhibition is likely at the level of polyadenylation, because the mutant SRp20 inhibits binding of CstF to the exon 4 poly(A) site. This is the first demonstration that an SR protein can influence alternative polyadenylation and suggests that this family of proteins may play a role in recognition of 3'-terminal exons and perhaps in the communication between polyadenylation and splicing.

Hammerhead ribozyme-mediated inactivation of mutant RET in medullary thyroid carcinoma.

Activating mutations of the RET proto-oncogene cause hereditary medullary thyroid carcinoma. To examine whether selective inactivation of mutant RET could prevent transformation, a hammerhead ribozyme was designed to cleave RET mRNA containing a transforming mutation of codon 634 TGC --> TAC (Cys634Tyr). In vitro RNA cleavage assay demonstrated that the ribozyme selectively cleaved RET RNA with a Cys634Tyr but not Cys634Arg or the normal sequence. Expression of ribozyme in NIH/3T3 cells prevented RET-mediated colony formation in soft agar. This inhibition required catalytically active ribozyme and was specific for the TAC mutation. Therefore, ribozymes designed to selectively target mutant RET RNA may provide an effective therapeutic in the treatment of this syndrome.

Duplication of the mutant RET allele in trisomy 10 or loss of the wild-type allele in multiple endocrine neoplasia type 2-associated pheochromocytomas.

Inherited mutations of the RET proto-oncogene are tumorigenic in patients with multiple endocrine neoplasia type 2 (MEN 2). However, it is not understood why only few of the affected cells in the target organs develop into tumors. Genetic analysis of nine pheochromocytomas from five unrelated patients with MEN 2 showed either duplication of the mutant RET allele in trisomy 10 or loss of the wild-type RET allele. Our results suggest a "second hit" causing a dominant effect of the mutant RET allele, through either duplication of the mutant allele or loss of the wild-type allele, as a possible mechanism for pheochromocytoma tumorigenesis in patients with MEN 2.

Allelic imbalance of the mutant and wild-type RET allele in MEN 2A-associated medullary thyroid carcinoma.

Germline mutations of the RET proto-oncogene are responsible for the familial tumor syndrome called multiple endocrine neoplasia type 2 (MEN 2) that includes medullary thyroid carcinoma (MTC). Although inherited mutations of RET lead to tumor formation in patients with MEN 2, it is not understood why only selected cells develop into tumors. We have recently shown that duplication of the mutated RET allele or loss of the wild-type allele might represent mechanisms of tumorigenesis in patients with MEN 2A-related pheochromocytoma. We now analysed 19 DNA samples of MTC (15 of which were non-microdissected, four of which were microdissected) from patients with MEN 2A. Using polymorphic marker and phosphorimage densitometry analyses, we found allelic imbalance of the mutated and wild-type RET allele in six of 19 DNA MTC samples. Of note, two of the four microdissected tumor DNA samples showed allelic imbalance of RET, whereas only four of the 15 non-microdissected MTC samples did. These results underscore the significance of microdissection in the analysis of tumor DNA. In our study, some of the non-microdissected tumor DNA samples may have failed to display allelic imbalance of RET, because of contamination of tumor DNA with nonneoplastic DNA or noninformative microsatellite marker analysis. Taken together, our results suggest allelic imbalance between mutated and wild-type RET as a possible mechanism for tumor formation in some patients with MEN 2A-related MTC.

The impact of gene mapping techniques on the management of multiple endocrine neoplasia type 2.

There has been sustained progress toward the identification of the gene for multiple endocrine neoplasia type 2. Closely linked and flanking DNA markers have been identified, and it is now possible to assign gene carrier status in informative families at risk with a >90% certainty by the use of molecular genetic techniques. Application of-these techniques, however, requires an understanding of their current limitations and caution in their use of clinical decision making.

The calcitonin exon and its flanking intronic sequences are sufficient for the regulation of human calcitonin/calcitonin gene-related peptide alternative RNA splicing.

The primary transcript of the calcitonin (CT)/calcitonin gene-related peptide (CGRP) is alternatively spliced in a cell-specific fashion to produce CT in thyroid C cells and CGRP in neuronal cells. The key step in this regulatory process is the recognition and inclusion of exon 4 to produce CT mRNA or nonrecognition and exclusion of exon 4 to produce CGRP mRNA. To determine whether inclusion/exclusion of CT exon is regulated independently of its position, we created a series of minigene constructs containing decreasing amounts of CT gene sequence. A human glioblastoma cell line, T98G, was tested and used as a CT exon exclusion cell line, while HeLa cells were used as a CT exon inclusion cell line. CT exon inclusion/exclusion was regulated when either the relative position of exon 4 within the CT gene was changed or when the exon with flanking sequence was inserted into a completely heterologous gene. Our results demonstrate that CT exon functions as a unit in a position-independent fashion in regulating its own inclusion/exclusion. We believe that the heterologous fusion gene containing only exon 4 and part of its flanking intron sequences will be useful for further defining the sequence elements involved in the regulation of CT/CGRP splicing.

Down-regulation of calcitonin gene transcription by vitamin D requires two widely separated enhancer sequences.

Transcription of the calcitonin (CT) gene is down-regulated by vitamin D in normal and transformed thyroid C cells. DNA transfer techniques have been previously used to map and characterize a cAMP-induced enhancer at nucleotides -255 to -129 and an enhancer of basal transcription at -1060 to -905 in the CT 5' flanking DNA. The same methods were used to identify a negative response element for vitamin D. Deletion mutants of a genomic fragment of CT extending from nucleotides -1460 to +90 were attached to a promoterless GH gene and transfected individually into the medullary thyroid carcinoma cell line TT. CT nucleotides -1460 to -129 induced significant basal transcription of the GH reporter gene in TT cells. Basal transcription was elevated 3-fold to 4-fold by treatment with cAMP analog. The biologically active metabolite of vitamin D3, 1,25-dihydroxyvitamin D3, had a minor (20%) inhibitory effect on basal transcription but inhibited more than 60% of the cAMP-induced transcription. We further investigated the cAMP-induced response and found that transcriptional activity of the downstream cAMP-induced enhancer was greatly synergized in the presence of the upstream enhancer of basal transcription. The latter enhancer contained three functional CANNTG sequences designated E1 (nucleotides -1060 to -1030), E2 (nucleotides -940 to -920), and E3 (nucleotides -920 to -900). E2 and E3 were essential for maximal cAMP-induced transcription. Detailed mapping of the vitamin D response showed that a minimum requirement for inhibition of the cAMP-induced enhancer by vitamin D was a sequence overlapping E3 (nucleotides -920 to -829). We conclude that a negative response element to vitamin D is located between nucleotides -920 and -829 in the CT 5' flanking DNA. It is possible that vitamin D inhibits transcription by interfering with the synergistic interaction between the cAMP-induced enhancer and the enhancer of basal transcription.

Transcription of the human calcitonin gene is mediated by a C cell-specific enhancer containing E-box-like elements.

The calcitonin (CT) gene is expressed normally in thyroidal C-cells and in a restricted population of cells in the central and peripheral nerve system. To define the cis-elements within the 5'-flanking DNA of the human CT gene which mediate this cell-specific expression, we used DNA transfer techniques and a transient transfection approach. We found that a DNA sequence located between -1290 and -820 of the CT 5'-flanking DNA functioned as an enhancer of basal transcription in C-cells (from medullary thyroid carcinoma) but not in rat glioma (C6), hamster insulinoma (HIT), fibroblasts (3T3), or epithelial cells (HeLa and CV1). Further mapping revealed the presence of at least two elements within the enhancer region; an upstream element (USE, located between -1060 and -1030) which could not function independently but its removal caused 70-80% loss of enhancer activity and a downstream element (DSE, located at -1033 to -920) which functioned independently as a cell-specific enhancer but with reduced activity. The binding pattern of nuclear proteins from C-cells to the enhancer elements was studied by an electrophoretic mobility shift assay. A protein-DNA complex was formed with the USE which could be competed, specifically, by an oligonucleotide containing the microE2 motif of the immunoglobulin gene enhancer. A similar complex was formed with the DSE fragment. Nuclear proteins from HeLa cells failed to form complexes with USE. Moreover, the binding pattern of proteins derived from HeLa cells to DSE was different from that of C-cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Cell type-specific regulation of transcription by cyclic adenosine 3,'5'-monophosphate-responsive elements within the calcitonin promoter.

A cAMP-induced enhancer was previously mapped to nucleotides -255 to -85 of the calcitonin (CT) gene 5'-flanking DNA. To determine the functional cis-acting elements within this region, we transfected medullary thyroid carcinoma (MTC) cells with CT 5'-flanking DNA/GH fusion genes containing potential cAMP-responsive elements and assessed their transcriptional activities with and without cAMP. In CT-expressing MTC cells (the TT line), we identified by deletions and point mutations three transcriptionally active motifs: a cAMP-responsive element (CRE), TGACGTCA, at -253 to -246, and a hybrid site containing a CRE-like element (CREL; TGACCTCA, -169 to -162) adjacent to an equally transcriptionally active octamer (O) sequence (ATG-CAAAT, -161 to -154). These three motifs acted synergistically and their transcriptional activity was completely dependent on cAMP. In HeLa cells their synergistic activity was more constitutive than cAMP induced, whereas in CT-negative MTC cells (the RO-D81-1 line) these motifs were inactive. Gel mobility shift assays with antibodies against CRE-binding protein (CREB) and activating transcription factor 1 (ATF-1) showed that both CREB and ATF-1 interacted with the CRE in MTC cells, whereas in HeLa cells only ATF-1 bound to the CRE. Specific binding to the CREL/O motif was detectable in extracts from tumors induced by injection of TT cells but not in extracts from any of the three cultured cell lines. We conclude that cAMP-induced transcription of the CT gene is modulated in a cell-specific manner by the CRE and the CREL/O elements.

1,25-Dihydroxyvitamin D3 silences 3',5'-cyclic adenosine monophosphate enhancement of somatostatin gene transcription in human thyroid C cells.

Treatment of the somatostatin (SRIF)-producing TT cell line with 1,25 dihydroxyvitamin D3 (1,25 D3) lowered intracellular SRIF mRNA and peptide concentration. In separate experiments, the cAMP analog 8-(4-chlorophenylthio)-cAMP stimulated a rapid increase in SRIF mRNA content of the TT cells and SRIF peptide secretion. To determine whether 1,25 D3 could inhibit either the transcriptional or secretory effects of cAMP, TT cells were pretreated with 1,25 D3 for 4 days followed by treatment with the cAMP analog. Pretreatment with 1,25 D3 inhibited the cAMP-mediated increase of SRIF mRNA, but had no effect on the secretory response. We conclude that the ability of 1,25 D3 to silence SRIF gene expression is more potent than cAMP enhancer activity but that 1,25 D3 has no effect on that portion of the cAMP-dependent pathway which regulates peptide secretion.

Calcitonin exon sequences influence alternative RNA processing.

The pre-mRNA encoding calcitonin (CT) and CT gene-related peptide (CGRP) is differentially processed in a tissue-specific fashion to include exon 4 (which encodes CT) or exclude this exon and splice to exon 5 (which encodes CGRP). We have used a CT-specific in vitro RNA-processing system to identify cis-acting sequences required to prevent splicing to exon 5. Deletion mapping demonstrated the presence of an element within the first 45 nucleotides of the CT-specific exon 4 that was required to suppress splicing to the CGRP-specific exon 5. This element was able to function in a completely heterologous system to suppress splicing when the CGRP exon was replaced with a constitutive viral exon. The element was unable to suppress splicing in the absence of a proximal CT-specific 3' splice site. Our results suggest that CT-specific splicing requires assisted recognition of its 3' splice site.

Streptozocin-treated Verner-Morrison Syndrome: plasma vasoactive intestinal peptide and tumor responses.

A patient with watery diarrhea, hypokalemia, hypochlorhydria, and a non-beta islet cell carcinoma of the pancreas (Verner-Morrison syndrome) was found to have an elevated vasoactive intestinal peptide (VIP) concentration in the plasma as well as in the tumor. Treatment with streptozocin resulted in a dramatic subjective and objective tumor response in this patient. Plasma VIP concentration fell into the normal range after four courses of treatment, diarrhea ceased after the third course of therapy, and measurable tumor mass markedly decreased during that same period of time. The patient remains in clinical remission with no evidence of tumor regrowth 18 months after the beginning of treatment. In this patient, plasma VIP measurements were an excellent marker of tumor activity and correlated well with objective disease measurements and clinical response.