THE JEREMIAH METZGER LECTURE: A BRIEF HISTORY OF EUGENICS IN AMERICA: IMPLICATIONS FOR MEDICINE IN THE 21ST CENTURY.

In the first half of the 20th century, the US was swept up in a multifaceted movement to enhance the genetic makeup of the country's population. This eugenics movement, based on flawed scientific principles promulgated by Galton in the UK and Davenport in the US included legally mandated compulsory sterilization in 27 states in the US and sharply restricted immigration from many parts of the world. Compulsory sterilization legislation was upheld by the Supreme Court in 1927. The American eugenics movement was a model for the compulsory sterilization implemented by the Nazis after they took power in Germany in 1933. The movement waned in America only following World War II when the US public became aware of the full extent of the Nazi Aryan racial superiority program. With the advent of major advances in molecular and cellular biology that are already being applied to clinical medicine in the 21st century, we have entered a new eugenics era. It is critical that we learn the lessons of our earlier eugenics movement if we are to avoid making the same flawed decisions now.

A double negative: inhibition of hepatic Gi signaling improves glucose homeostasis.

Hepatic glucose production (HGP) is a key determinant of glucose homeostasis. Glucagon binding to its cognate seven-transmembrane Gs-coupled receptor in hepatocytes stimulates cAMP production, resulting in increased HGP. In this issue of the JCI, Rossi and colleagues tested the hypothesis that activation of hepatic Gi-coupled receptors, which should inhibit cAMP production, would oppose the cAMP-inducing action of glucagon and thereby decrease HGP. Surprisingly, however, the opposite occurred: activation of Gi signaling increased HGP via a novel mechanism, while inhibition of Gi signaling reduced HGP. These results define a new physiologic role for hepatic Gi signaling and identify a potential therapeutic target for HGP regulation.

Allosteric Modulation of the Calcium-sensing Receptor Rectifies Signaling Abnormalities Associated with G-protein α-11 Mutations Causing Hypercalcemic and Hypocalcemic Disorders.

Germline loss- and gain-of-function mutations of G-protein α-11 (Gα11), which couples the calcium-sensing receptor (CaSR) to intracellular calcium (Ca(2+) i) signaling, lead to familial hypocalciuric hypercalcemia type 2 (FHH2) and autosomal dominant hypocalcemia type 2 (ADH2), respectively, whereas somatic Gα11 mutations mediate uveal melanoma development by constitutively up-regulating MAPK signaling. Cinacalcet and NPS-2143 are allosteric CaSR activators and inactivators, respectively, that ameliorate signaling disturbances associated with CaSR mutations, but their potential to modulate abnormalities of the downstream Gα11 protein is unknown. This study investigated whether cinacalcet and NPS-2143 may rectify Ca(2+) i alterations associated with FHH2- and ADH2-causing Gα11 mutations, and evaluated the influence of germline gain-of-function Gα11 mutations on MAPK signaling by measuring ERK phosphorylation, and assessed the effect of NPS-2143 on a uveal melanoma Gα11 mutant. WT and mutant Gα11 proteins causing FHH2, ADH2 or uveal melanoma were transfected in CaSR-expressing HEK293 cells, and Ca(2+) i and ERK phosphorylation responses measured by flow-cytometry and Alphascreen immunoassay following exposure to extracellular Ca(2+) (Ca(2+) o) and allosteric modulators. Cinacalcet and NPS-2143 rectified the Ca(2+) i responses of FHH2- and ADH2-associated Gα11 loss- and gain-of-function mutations, respectively. ADH2-causing Gα11 mutations were demonstrated not to be constitutively activating and induced ERK phosphorylation following Ca(2+) o stimulation only. The increased ERK phosphorylation associated with ADH2 and uveal melanoma mutants was rectified by NPS-2143. These findings demonstrate that CaSR-targeted compounds can rectify signaling disturbances caused by germline and somatic Gα11 mutations, which respectively lead to calcium disorders and tumorigenesis; and that ADH2-causing Gα11 mutations induce non-constitutive alterations in MAPK signaling.

The Calcilytic Agent NPS 2143 Rectifies Hypocalcemia in a Mouse Model With an Activating Calcium-Sensing Receptor (CaSR) Mutation: Relevance to Autosomal Dominant Hypocalcemia Type 1 (ADH1).

Autosomal dominant hypocalcemia type 1 (ADH1) is caused by germline gain-of-function mutations of the calcium-sensing receptor (CaSR) and may lead to symptomatic hypocalcemia, inappropriately low serum PTH concentrations and hypercalciuria. Negative allosteric CaSR modulators, known as calcilytics, have been shown to normalize the gain-of-function associated with ADH-causing CaSR mutations in vitro and represent a potential targeted therapy for ADH1. However, the effectiveness of calcilytic drugs for the treatment of ADH1-associated hypocalcemia remains to be established. We have investigated NPS 2143, a calcilytic compound, for the treatment of ADH1 by in vitro and in vivo studies involving a mouse model, known as Nuf, which harbors a gain-of-function CaSR mutation, Leu723Gln. Wild-type (Leu723) and Nuf mutant (Gln723) CaSRs were expressed in HEK293 cells, and the effect of NPS 2143 on their intracellular calcium responses was determined by flow cytometry. NPS 2143 was also administered as a single ip bolus to wild-type and Nuf mice and plasma concentrations of calcium and PTH, and urinary calcium excretion measured. In vitro administration of NPS 2143 decreased the intracellular calcium responses of HEK293 cells expressing the mutant Gln723 CaSR in a dose-dependent manner, thereby rectifying the gain-of-function associated with the Nuf mouse CaSR mutation. Intraperitoneal injection of NPS 2143 in Nuf mice led to significant increases in plasma calcium and PTH without elevating urinary calcium excretion. These studies of a mouse model with an activating CaSR mutation demonstrate NPS 2143 to normalize the gain-of-function causing ADH1 and improve the hypocalcemia associated with this disorder.

Research in academic medical centers: two threats to sustainable support.

Reductions in federal support and clinical revenue jeopardize biomedical research and, in turn, clinical medicine.

The stem cell wars: a dispatch from the front.

The development of human embryonic stem cell (hESC) lines in 1998 offered the prospect of a new era of regenerative medicine in which cell therapy might cure intractable diseases such as type 1 diabetes, Parkinson's disease, and spinal cord injury. The Bush Administration decision in 2001 to restrict federal funding of hESC research touched off a controversy that continues to the present. One response to the Bush policy was establishment of state stem cell research funding programs, notably the California Institute of Regenerative Medicine (CIRM). As Director of the National Institute of Diabetes, Digestive, and Kidney Diseases (NIDDK) and Vice Chair of the National Institutes of Health (NIH) Stem Cell Task Force, and now as a member of the Empire State Stem Cell Funding Board and member of an Institute of Medicine (IOM) committee charged with evaluation of the CIRM, I have had the opportunity to gain a first-hand perspective of the field. Here I present my impressions of the legal and science policy debates and selectively summarize research progress toward the hoped-for cures.

'Personalized medicine' to identify genetic risks for type 2 diabetes and focus prevention: can it fulfill its promise?

Public health measures are required to address the worldwide increase in type 2 diabetes. Proponents of personalized medicine predict a future in which disease treatment and, more important, prevention will be tailored to high-risk individuals rather than populations and will be based on genetic and other new biomarker tests. Accurate biomarker tests to identify people at risk for diabetes could allow more-targeted and perhaps individualized prevention efforts. DNA variants conferring higher risk for type 2 diabetes have been identified. However, these account for only a small fraction of genetic risk, which limits their practical predictive value. Nor has identification of these variants yet led to new, individualized prevention methods. Further research is needed to identify genomic and other types of biomarkers that could accurately predict risk and facilitate targeted prevention.

Future diagnostic and therapeutic trends in endocrine cancers.

Current treatment of endocrine cancers relies primarily on surgical resection, which is generally effective only for localized disease. Radioactive iodine treatment is an important modality for those thyroid cancers that maintain the ability to take up iodine. For endocrine cancers that are no longer localized, current modes of therapy, including various combinations of chemotherapy and radiation, are inadequate, posing a major challenge to ongoing research to develop more effective methods for diagnosis and treatment. In this article, we offer some predictions of future trends in the diagnosis and treatment of endocrine cancers. Following a general introduction, we focus on thyroid cancer as a paradigm for what we may expect in future developments, and then add selected comments relevant to parathyroid, adrenocortical, and gastrointestinal and pancreatic neuroendocrine tumors. Rapid, inexpensive whole genome sequencing of both germline and tumor DNA, novel molecular and functional imaging, as well as new biomarkers are expected to enable more precise diagnosis, targeted therapy, and possibly prevention. Translating the coming wave of data on the molecular pathogenesis of endocrine cancers into practical diagnostic and treatment modalities will require new forms of collaboration between investigators, clinicians, and industry.

Commentary: new guidelines for NIH peer review: improving the system or undermining it?

The National Institutes of Health (NIH) peer review system has been viewed as the best way to guarantee the scientific independence of biomedical research in the United States, and it has been emulated internationally. The system, however, is subject to a variety of stresses, and these have always been exacerbated at times of flat NIH funding, as in the past five years. To address several of these stresses, NIH first conducted a "diagnostic self-study" of the peer review system and then implemented a number of changes. Costello, in a Perspective in this issue of Academic Medicine, argues that two of these changes, special consideration for new investigators and emphasis of the criterion of "innovation," undermine the stated goal of funding the "best science by the best scientists." In this commentary on Costello's Perspective article, the author examines the issue of NIH funding of new investigators from a historical perspective, in the context of overall NIH priority setting in resource allocation. The related issue of innovation as a criterion in NIH peer review is also addressed, and the commentary concludes with an affirmation of the need to measure outcomes in assessing the impact of changes in the NIH peer review system.

Recapitulation of pancreatic neuroendocrine tumors in human multiple endocrine neoplasia type I syndrome via Pdx1-directed inactivation of Men1.

Multiple endocrine neoplasia type 1 (MEN1) is an autosomal syndrome caused by mutations in the MEN1 tumor suppressor gene. Whereas the protein product of MEN1, menin, is ubiquitously expressed, somatic loss of the remaining wild-type MEN1 allele results in tumors primarily in parathyroid, pituitary, and endocrine pancreas. To understand the endocrine specificity of the MEN1 syndrome, we evaluated biallelic loss of Men1 by inactivating Men1 in pancreatic progenitor cells using the Cre-lox system. Men1 deletion in progenitor cells that differentiate into exocrine and endocrine pancreas did not affect normal pancreas morphogenesis and development. However, mice having homozygous inactivation of the Men1 in pancreas developed endocrine tumors with no exocrine tumor manifestation, recapitulating phenotypes seen in the MEN1 patients. In the absence of menin, the endocrine pancreas showed increase in cell proliferation, vascularity, and abnormal vascular structures; such changes were lacking in exocrine pancreas. Further analysis revealed that these endocrine manifestations were associated with up-regulation in vascular endothelial growth factor expression in both human and mouse MEN1 pancreatic endocrine tumors. Together, these data suggest the presence of cell-specific factors for menin and a permissive endocrine environment for MEN1 tumorigenesis in endocrine pancreas. Based on our analysis, we propose that menin's ability to maintain cellular and microenvironment integrity might explain the endocrine- restrictive nature of the MEN1 syndrome.

Parathyroid tumor development involves deregulation of homeobox genes.

Multiple endocrine neoplasia type 1 (MEN1) is an autosomal dominant syndrome caused by mutations in the MEN1 tumor suppressor gene. Loss of the functional second copy of the MEN1 gene causes individuals to develop multiple endocrine tumors, primarily affecting the parathyroid, pituitary, and pancreas. While it is clear that the protein encoded by MEN1, menin, suppresses endocrine tumors, its biochemical functions and direct downstream targets remain unclear. Recent studies have suggested that menin may act as a scaffold protein to coordinate gene transcription, and that menin is an oncogenic cofactor for homeobox (HOX) gene expression in hematopoietic cancer. The role of HOX genes in adult cell differentiation is still obscure, but growing evidence suggests that they may play important roles in the development of cancer. Therefore, we hypothesized that specific HOX genes were regulated by menin in parathyroid tumor development. Utilizing quantitative TaqMan RT-PCR, we compared expression profiles of the 39 HOX genes in human familial MEN1 (fMEN1) parathyroid tumors and sporadic parathyroid adenomas with normal samples. We identified a large set of 23 HOX genes whose deregulation is specific for fMEN1 parathyroid tumors, and only 5 HOX genes whose misexpression are specific for sporadic parathyroid tumor development. These findings provide the first evidence that loss of the MEN1 tumor suppressor gene is associated with deregulation of specific HOX gene expression in the development of familial human parathyroid tumors. Our results strongly reinforce the idea that abnormal expression of developmental HOX genes can be critical in human cancer progression.

Inherited endocrine diseases involving G proteins and G protein-coupled receptors.

Naturally occurring mutations in the G protein Gs-alpha subunit and in a number of G protein-coupled receptors (GPCRs) have been identified in human diseases. Loss-of-function mutations in GPCRs for various hormones lead to hormone resistance manifest as hypofunction of the gland expressing the affected GPCR. Conversely, GPCR gain-of-function mutations lead to hormone-independent activation and hyperfunction of the involved gland. Our laboratory has focused on the extracellular calcium-sensing GPCR (CaR) expressed primarily, but not exclusively, in parathyroid glands and kidney. Loss-of-function CaR mutations lead to a form of hyperparathyroidism, an apparent exception to the general pattern described above, but in fact reflecting resistance to the normal inhibition of parathyroid hormone secretion by the 'hormone' agonist, extracellular Ca2+. CaR gain-of function-mutations cause autosomal dominant hypocalcemia due to activation of the receptor at subphysiologic concentrations of serum Ca2+, leading to 'inappropriate' inhibition of parathyroid hormone secretion. I will describe our recent work that helps inform design of novel therapeutics targeting this important GPCR.

Structure and function of the human calcium-sensing receptor: insights from natural and engineered mutations and allosteric modulators.

The human extracellular Ca(2+)-sensing receptor (CaR), a member of the G protein-coupled receptor family 3, plays a key role in the regulation of extracellular calcium homeostasis. It is one of just a few G protein-coupled receptors with a large number of naturally occurring mutations identified in patients. In contrast to the small sizes of its agonists, this large dimeric receptor consists of domains with topologically distinctive orthosteric and allosteric sites. Information derived from studies of naturally occurring mutations, engineered mutations, allosteric modulators and crystal structures of the agonist-binding domain of homologous type 1 metabotropic glutamate receptor and G protein-coupled rhodopsin offers new insights into the structure and function of the CaR.

Distribution of menin-occupied regions in chromatin specifies a broad role of menin in transcriptional regulation.

Menin is the protein product of the MEN1 tumor-suppressor gene; one allele of MEN1 is inactivated in the germ line of patients with "multiple endocrine neoplasia type 1" (MEN1) cancer syndrome. Menin interacts with several proteins involved in transcriptional regulation. RNA expression analyses have identified several menin-regulated genes that could represent proximal or distal interaction sites for menin. This report presents a substantial and unbiased sampling of menin-occupied chromatin regions using Serial Analysis of Chromatin Occupancy; this method combines chromatin immuno-precipitation with Serial Analysis of Gene Expression. Hundreds of menin-occupied genomic sites were identified in promoter regions (32% of menin-occupied loci), near the 3' end of genes (14%), or inside genes (21%), extending other data about menin recruitments to many sites of transcriptional activity. A large number of menin-occupied sites (33%) were located outside known gene regions. Additional annotation of the human genome could help in identifying genes at these loci, or these might be gene-free regions of the genome where menin occupancy could play some structural or regulatory role. Menin occupancy at many intragenic positions distant from the core promoter reveals an unexpected type of menin target region at many loci in the genome. These unbiased data also suggest that menin could play a broad role in transcriptional regulation.

The parathyroid/pituitary variant of multiple endocrine neoplasia type 1 usually has causes other than p27Kip1 mutations.

CONTEXT: One variant of multiple endocrine neoplasia type 1 (MEN1) is defined by sporadic tumors of both the parathyroids and pituitary. The prevalence of identified MEN1 mutations in this variant is lower than in familial MEN1 (7% vs. 90%), suggesting different causes. Recently, one case of this variant had a germline mutation of p27(Kip1)/CDKN1B. OBJECTIVE: The objective was to test p27 in germline DNA from cases with tumors of both the parathyroids and pituitary. DESIGN: Medical record review and sequence analysis in DNA were performed. SETTING: This study involved an inpatient and outpatient referral program for cases of endocrine tumors. PATIENTS: Sixteen index cases had sporadic tumors of two organs, both the parathyroids and the pituitary. There were 18 additional index cases with related features of familial tumors. Five subjects were normal controls. No case had an identified MEN1 mutation. INTERVENTIONS: Clinical status of endocrine tumors was tabulated. Sequencing of germline DNA from index cases and control cases for the p27 gene was performed by PCR. MAIN OUTCOME MEASURES: Endocrine tumor types and their expressions were measured, as were sequence changes in the p27 gene. RESULTS: Tumor features were documented in index cases and families. One p27 germline single nucleotide change was identified. This predicted a silent substitution of Thr142Thr. Furthermore, there was a normal prevalence of heterozygosity for a common p27 polymorphism, making a large p27 deletion unlikely in all or most of these cases. CONCLUSIONS: The MEN1 variant with sporadic parathyroid tumors, sporadic pituitary tumor, and no identified MEN1 mutation is usually not caused by p27 germline mutations. It is usually caused by as yet unknown process(es).

Functional effects of monoclonal antibodies to the purified amino-terminal extracellular domain of the human Ca(2+) receptor.

UNLABELLED: We generated three functionally unique monoclonal antibodies to the purified human CaR extracellular domain. Flow cytometry studies of chimeric receptors localized their epitopes to lobe 2 of the VFT domain. These results lead us to propose a mechanism for the functional effects of these antibodies. INTRODUCTION: The human Ca(2+) receptor (CaR), which plays a central role in the regulation of [Ca(2+)](0) homeostasis, has a distinctively large extracellular domain that consists of a bilobed Venus flytrap (VFT) domain, involved in agonist binding, and a cysteine-rich domain. Functional antibodies that specifically bind to this domain would have therapeutic potential and could be used as a tool to gain insights into receptor activation as well. MATERIALS AND METHODS: We generated three monoclonal antibodies (mAbs), 7F8, 5C8, and 1A8, to the purified human CaR extracellular domain. Functional characterization of these antibodies included Ca(2+) stimulation of phosphoinositide hydrolysis to examine effects of intact or protease digested antibodies on sensitivity of the receptor to extracellular Ca(2+) and flow cytometry assay of binding of the antibodies to HEK-293 cells expressing chimeric receptors to map antibody epitopes. RESULTS: We found these mAbs specifically recognize native but not denatured human CaR or homologous native Fugu CaR. Sensitivity of the human CaR to extracellular calcium was increased by binding of 5C8 but decreased by binding of 1A8. A chimeric receptor FCFCF, with lobe 2 region of the human CaR VFT domain in the Fugu CaR backbone, bound all three mAbs, and the sensitivity of this chimeric CaR to extracellular Ca(2+) was also increased by binding of 5C8 and decreased by binding of 1A8. CONCLUSIONS: The epitopes of these mAbs reside in the lobe 2 region of the human CaR VFT domain. 5C8 might activate the receptor by facilitating closure and/or rotation of the VFT domains on agonist binding, whereas 1A8 might inhibit the receptor by impeding such agonist-induced conformational changes. Recombinant antibodies with antigen binding domains of 5C8 and 1A8 could be useful in the treatment of hyperparathyroidism and osteoporosis, respectively.

Parathyroid-specific double knockout of Gq and G11 alpha-subunits leads to a phenotype resembling germline knockout of the extracellular Ca2+ -sensing receptor.

Germline knockout of the extracellular Ca2+ -sensing receptor (CaR) leads to a phenotype that includes severe hypercalcemia, hyperparathyroidism, relative hypocalciuria, skeletal abnormalities, retarded growth, and early postnatal death. To investigate the role of heterotrimeric G proteins in CaR signaling, we used cre/lox technology to delete the respective alpha-subunits of Gq and G11 selectively in parathyroid cells. Mice that were PTH-Cre(+/-); Gnaq(flox/flox); Gna11(-/-) (PTH-Galphaq/Galpha11 -double knockouts) were viable, but showed all the features of germline knockout of the CaR except hypocalcuria. Our results demonstrate the critical role of both Gq and G11 in mediating inhibition of PTH secretion by extracellular Ca2+.

A missense mutation in the seven-transmembrane domain of the human Ca2+ receptor converts a negative allosteric modulator into a positive allosteric modulator.

G protein-coupled receptors (GPCRs) are the most common targets of drug action. Allosteric modulators bind to the seven-transmembrane domain of family 3 GPCRs and offer enhanced selectivity over orthosteric ligands that bind to the large extracellular N terminus. We characterize a novel negative allosteric modulator of the human Ca(2+) receptor, Compound 1, that retains activity against the E837A mutant that lacks a response to previously described positive and negative modulators. A related compound, JKJ05, acts as a negative allosteric modulator on the wild type receptor but as a positive modulator on the E837A mutant receptor. This positive modulation critically depends on the primary amine in JKJ05, which appears to interact with acidic residue Glu(767) in our model of the seven-transmembrane domain of the receptor. Our results suggest the need for identification of possible genetic variation in the allosteric site of therapeutically targeted GPCRs.