This proof-of-concept analysis showed that circulating tumor DNA is an informative, inherently specific, and highly sensitive biomarker of metastatic breast cancer. (Funded by Cancer Research UK and others.).

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Sum m a r yThyroid hormones exert their effects through alpha (TRα1) and beta (TRβ1 and TRβ2) receptors. Here we describe a child with classic features of hypothyroidism (growth retardation, developmental retardation, skeletal dysplasia, and severe constipation) but only borderline-abnormal thyroid hormone levels. Using wholeexome sequencing, we identified a de novo heterozygous nonsense mutation in a gene encoding thyroid hormone receptor alpha (THRA) and generating a mutant protein that inhibits wild-type receptor action in a dominant negative manner. Our observations are consistent with defective human TRα-mediated thyroid hormone resistance and substantiate the concept of hormone action through distinct receptor subtypes in different target tissues.T hyroid hormones have diverse actions, which include regulation of skeletal growth, maturation of the central nervous system, cardiac and gastrointestinal function, and energy homeostasis. In addition, thyroid hormones control their own production by feedback inhibition of hypothalamic thyrotropinreleasing hormone and pituitary thyroid-stimulating hormone, which direct their synthesis or release. These physiological effects are principally mediated by hormone action through nuclear receptor proteins that act as ligand-inducible transcription factors and either positively or negatively regulate the expression of target genes in different tissues in a hormone-dependent manner.The receptors are encoded by two genes (THRA and THRB), each of which undergoes alternate splicing to generate receptor subtypes (TRα1, TRβ1, and TRβ2), with differing tissue distributions. TRα1 is the predominant subtype in bone, the gastrointestinal tract, cardiac and skeletal muscle, and the central nervous system; TRβ1 is most abundant in the liver and kidney; and TRβ2 is more discretely expressed in the hypothalamus, pituitary, cochlea, and retina. 1 In the absence of hormone, thyroid receptors that are not bound to ligands repress or silence targetgene transcription by recruiting multiprotein complexes containing corepressors (e.g., nuclear receptor corepressor and silencing mediator of retinoic acid and thyroid hormone receptor), with histone deacetylase activity; triiodothyronine occupancyThe New England Journal of Medicine Downloaded from nejm.org on May 10, 2018. For personal use only. No other uses without permission.

Cpn60 was labeled with pyrene maleimide in order to follow structural rearrangements in the protein triggered by the binding of nucleotides and cpn10. The conjugate binds ATP, AMP-PNP, and ADP(P(i)) with pyrene fluorescence enhancements of 60%, 60%, and 15%, respectively. In each case, binding is cooperative with half-saturation (K1/2) occurring at 10 microM, 290 microM, and 2500 microM and Hill constants (nH) of 4, 3, and 3, respectively. Inclusion of the co-protein, cpn10, tightens the binding of ATP, AMP-PNP, and ADP(P(i)) to give K1/2 values of 6 microM, 100 microM, and < 0.07 microM, respectively, and cooperativity is increased. Titration of the cpn60/ADP (14-mer) complex with cpn10 (7-mer) gives a stoichiometry of 14:7 with respect to subunits, confirming the molecular asymmetry shown by electron microscopy. Transient kinetics demonstrate that ATP initially forms a weak collision complex with cpn60 (Kd = 4 mM) which isomerizes to the strongly binding state at a rate of 180 s-1. We suggest that the slow structural rearrangement driven by ATP binding is the same event which lowers the affinity of the chaperonin for protein substrates; a suggestion reinforced by the loss of AMP-PNP binding affinity in the presence of an unstructured polypeptide. As such, this rearrangement of cpn60 is analogous to a force-generating step in energy transduction. Measurements of ATP hydrolysis (pH 7.5, 25 degrees C) show that it is slow (0.04 s-1) compared both with the structural rearrangement and with the dissociation of products. This defines the steady-state complex as cpn60/ATP, a form of the chaperonin which binds substrate proteins weakly. The rate of hydrolysis of ATP is stimulated 20-fold upon binding unfolded lactate dehydrogenase, and the yield of folded enzyme is increased even in the absence of cpn10. Addition of this co-protein inhibits hydrolysis on only half of the sites in cpn60 and leads to a faster release of folded LDH. A mechanism for the action of chaperonins is proposed which depends upon cpn60 being cycled between states which have, alternately, low and high affinity for unfolded proteins. This cycle is driven by the binding and hydrolysis of ATP.

The importance of the nuclear receptor peroxisome proliferator-activated receptor-␥ (PPAR␥) in regulating insulin resistance and blood pressure has been demonstrated in families with loss of function mutations. Gain of function mutations has been associated with severe obesity. However, previous population studies of the common variant Pro12Ala have produced conflicting results. As it is likely that the natural ligands for this receptor may include fatty acids, we hypothesized that the effect of this common variant may be altered by the character of the diet, particularly the ratio of dietary polyunsaturated fat to saturated fat (P:S ratio). We studied 592 nondiabetic participants in an ongoing population-based cohort study who were genotyped for the Pro12Ala polymorphism in the PPAR ␥2 isoform. As the Ala homozygotes were uncommon (2.0%), all analyses were conducted comparing Pro homozygotes (79.1%) to Ala allele carriers. There was no difference in fasting insulin concentration or BMI between Ala allele carriers and Pro homozygotes. The fasting insulin concentration was negatively associated with the P:S ratio (P ‫؍‬ 0.0119) after adjustment for age and sex, and a strong interaction was evident between the P:S ratio and the Pro12Ala polymorphism for both BMI (P ‫؍‬ 0.0038) and fasting insulin (P ‫؍‬ 0.0097). The data suggest that when the dietary P:S ratio is low, the BMI in Ala carriers is greater than that in Pro homozygotes, but when the dietary ratio is high, the opposite is seen. This gene-nutrient interaction emphasizes the difficulty of examining the effect of common polymorphisms in the absence of data on nongenetic exposures, and may explain the heterogeneity of findings in previous studies. Diabetes 50: 686 -689, 2001 A complete understanding of the etiology of common complex diseases such as diabetes, ischemic heart disease, and obesity will require exploration of the interactions between environmental factors and common genetic variants (1). To date, few examples of such interactions have been described. We present evidence for an environment-gene interaction influencing BMI and insulin sensitivity involving the nuclear receptor peroxisome proliferator-activated receptor-␥ (PPAR␥) and the pattern of dietary fat intake.A critical role for PPAR␥ in the development of mammalian adipose tissue has been confirmed by the absence of adipose tissue in PPAR␥ null murine embryos (2). Additionally, the importance of PPAR␥ in the control of human insulin sensitivity and blood pressure has recently been established by the finding of severe insulin-resistant diabetes and early-onset hypertension in two families with loss of function mutations in this gene (3). A gain of function mutation in PPAR␥ has also been reported in three subjects with severe obesity (4). Several studies have examined the relationship between a common variant (Pro12Ala) in the PPAR␥ isoform (5) and metabolic variables (6 -11). In terms of adiposity, studies have had variable results with some reporting increased (6), some decreased (7,8...

Selenium, a trace element that is fundamental to human health, is incorporated into some proteins as selenocysteine (Sec), generating a family of selenoproteins. Sec incorporation is mediated by a multiprotein complex that includes Sec insertion sequence-binding protein 2 (SECISBP2; also known as SBP2). Here, we describe subjects with compound heterozygous defects in the SECISBP2 gene. These individuals have reduced synthesis of most of the 25 known human selenoproteins, resulting in a complex phenotype. Azoospermia, with failure of the latter stages of spermatogenesis, was associated with a lack of testis-enriched selenoproteins. An axial muscular dystrophy was also present, with features similar to myopathies caused by mutations in selenoprotein N (SEPN1). Cutaneous deficiencies of antioxidant selenoenzymes, increased cellular ROS, and susceptibility to ultraviolet radiation-induced oxidative damage may mediate the observed photosensitivity. Reduced levels of selenoproteins in peripheral blood cells were associated with impaired T lymphocyte proliferation, abnormal mononuclear cell cytokine secretion, and telomere shortening. Paradoxically, raised ROS in affected subjects was associated with enhanced systemic and cellular insulin sensitivity, similar to findings in mice lacking the antioxidant selenoenzyme glutathione peroxidase 1 (GPx1). Thus, mutation of SECISBP2 is associated with a multisystem disorder with defective biosynthesis of many selenoproteins, highlighting their role in diverse biological processes.

Current serum biomarkers for diagnosis and monitoring of malignant germ cell tumors (GCTs) show limited sensitivity and specificity. We previously observed that microRNAs of the miR-371∼373 and miR-302 clusters are overexpressed in all malignant GCTs, regardless of patient age, histologic subtype, or anatomic site, but are not reported to be coordinately up-regulated in other tumor types or disease states. Herein we show that levels of all 8 main members of the miR-371∼373 and miR-302 clusters were elevated in the serum of a 4-year-old boy at the time of diagnosis of yolk sac tumor. Levels returned to normal during an uneventful clinical follow-up, with kinetics similar to those of the conventional marker α-fetoprotein. We describe in detail the multiplex polymerase chain reaction protocol used to quantify serum microRNA levels, which is highly robust and reproducible. Our study indicates that miR-371∼373 and miR-302 cluster microRNAs are promising candidate biomarkers for improving disease monitoring (and potentially diagnosis) in malignant GCTs.

Background and Purpose-Stroke leads to a reduction in bone mineral density, altered calcium homeostasis, and an increase in hip fractures. Vitamin D deficiency is well documented in long-term stroke survivors and is associated with post-stroke hip fractures. Less is known regarding levels in acute stroke. Methods-We compared the serum 25-dihydroxyvitamin D levels of 44 patients admitted to an acute stroke unit with first-ever stroke with results obtained by measuring 96 healthy ambulant elderly subjects every 2 months for 1 year. Statistical Z scores of serum vitamin D were then calculated after seasonal adjustment for the month of sampling. Results-The mean Z score of vitamin D in acute stroke was Ϫ1.4 SD units (95% CI, Ϫ1.7, Ϫ1.1), with 77% of patients falling in the insufficient range. Conclusions-Reduced