Genetic Variants Associated with Serum Thyroid Stimulating Hormone (TSH) Levels in European Americans and African Americans from the eMERGE Network

Thyroid stimulating hormone (TSH) hormone levels are normally tightly regulated within an individual; thus, relatively small variations may indicate thyroid disease. Genome-wide association studies (GWAS) have identified variants in PDE8B and FOXE1 that are associated with TSH levels. However, prior studies lacked racial/ethnic diversity, limiting the generalization of these findings to individuals of non-European ethnicities. The Electronic Medical Records and Genomics (eMERGE) Network is a collaboration across institutions with biobanks linked to electronic medical records (EMRs). The eMERGE Network uses EMR-derived phenotypes to perform GWAS in diverse populations for a variety of phenotypes. In this report, we identified serum TSH levels from 4,501 European American and 351 African American euthyroid individuals in the eMERGE Network with existing GWAS data. Tests of association were performed using linear regression and adjusted for age, sex, body mass index (BMI), and principal components, assuming an additive genetic model. Our results replicate the known association of PDE8B with serum TSH levels in European Americans (rs2046045 p = 1.85×10⁻¹⁷, β = 0.09). FOXE1 variants, associated with hypothyroidism, were not genome-wide significant (rs10759944: p = 1.08×10⁻⁶, β = −0.05). No SNPs reached genome-wide significance in African Americans. However, multiple known associations with TSH levels in European ancestry were nominally significant in African Americans, including PDE8B (rs2046045 p = 0.03, β = −0.09), VEGFA (rs11755845 p = 0.01, β = −0.13), and NFIA (rs334699 p = 1.50×10⁻³, β = −0.17). We found little evidence that SNPs previously associated with other thyroid-related disorders were associated with serum TSH levels in this study. These results support the previously reported association between PDE8B and serum TSH levels in European Americans and emphasize the need for additional genetic studies in more diverse populations.     

Hypoxia Enhances the Antiglioma Cytotoxicity of B10, a Glycosylated Derivative of Betulinic Acid

B10 is a glycosylated derivative of betulinic acid with promising activity against glioma cells. Lysosomal cell death pathways appear to be essential for its cytotoxicity. We investigated the influence of hypoxia, nutrient deprivation and current standard therapies on B10 cytotoxicity. The human glioma cell lines LN-308 and LNT-229 were exposed to B10 alone or together with irradiation, temozolomide, nutrient deprivation or hypoxia. Cell growth and viability were evaluated by crystal violet staining, clonogenicity assays, propidium iodide uptake and LDH release assays. Cell death was examined using an inhibitor of lysosomal acidification (bafilomycin A1), a cathepsin inhibitor (CA074-Me) and a short-hairpin RNA targeting cathepsin B. Hypoxia substantially enhanced B10-induced cell death. This effect was sensitive to bafilomycin A1 and thus dependent on hypoxia-induced lysosomal acidification. Cathepsin B appeared to mediate cell death because either the inhibitor CA074-Me or cathepsin B gene silencing rescued glioma cells from B10 toxicity under hypoxia. B10 is a novel antitumor agent with substantially enhanced cytotoxicity under hypoxia conferred by increased lysosomal cell death pathway activation. Given the importance of hypoxia for therapy resistance, malignant progression, and as a result of antiangiogenic therapies, B10 might be a promising strategy for hypoxic tumors like malignant glioma.     

An Attenuated Herpes Simplex Virus Type 1 (HSV1) Encoding the HIV-1 Tat Protein Protects Mice from a Deadly Mucosal HSV1 Challenge

Herpes simplex virus types 1 and 2 (HSV1 and HSV2) are common infectious agents in both industrialized and developing countries. They cause recurrent asymptomatic and/or symptomatic infections, and life-threatening diseases and death in newborns and immunocompromised patients. Current treatment for HSV relies on antiviral medications, which can halt the symptomatic diseases but cannot prevent the shedding that occurs in asymptomatic patients or, consequently, the spread of the viruses. Therefore, prevention rather than treatment of HSV infections has long been an area of intense research, but thus far effective anti-HSV vaccines still remain elusive. One of the key hurdles to overcome in anti-HSV vaccine development is the identification and effective use of strategies that promote the emergence of Th1-type immune responses against a wide range of epitopes involved in the control of viral replication. Since the HIV1 Tat protein has several immunomodulatory activities and increases CTL recognition of dominant and subdominant epitopes of heterologous antigens, we generated and assayed a recombinant attenuated replication-competent HSV1 vector containing the tat gene (HSV1-Tat). In this proof-of-concept study we show that immunization with this vector conferred protection in 100% of mice challenged intravaginally with a lethal dose of wild-type HSV1. We demonstrate that the presence of Tat within the recombinant virus increased and broadened Th1-like and CTL responses against HSV-derived T-cell epitopes and elicited in most immunized mice detectable IgG responses. In sharp contrast, a similarly attenuated HSV1 recombinant vector without Tat (HSV1-LacZ), induced low and different T cell responses, no measurable antibody responses and did not protect mice against the wild-type HSV1 challenge. These findings strongly suggest that recombinant HSV1 vectors expressing Tat merit further investigation for their potential to prevent and/or contain HSV1 infection and dissemination.     

Targeted sequencing of a complex locus within a polyploid genome using reduced representation libraries

Apospory is a form of gametophytic apomixis in which embryos develop from unreduced embryo sacs derived from aposporous initials formed from nucellar cells of ovules to produce offspring genetically identical to the female plant. Apospory in Pennisetum squamulatum (8X) and Cenchrus ciliaris (4X) is a dominant trait controlled by a physically large, hemizygous, heterochromatic chromosomal block called the apospory-specific genomic region (ASGR). Both apomictic species are polyploid, with genome sizes estimated at 2600 to 3000 Mbp for C. ciliaris and 9400 to 10,300 Mbp for P. squamulatum. A study was conducted to determine whether duplex-specific nuclease (DSN) normalization of DNA from apomictic and sexual genotypes would reduce repetitive sequences and allow bioinformatic analysis to predict sequence contigs derived from the ASGR. DSN libraries from four genotypes were sequenced using Illumina^® HiSeq 2000 technology. 39 out of 44 tested sequence characterized amplified region (SCAR) markers from in silico predicted ASGR-specific contigs were mapped to the ASGR in a Pennisetum F₁ mapping population. Eighteen SCARs showed apomict-specific amplification in C. ciliaris. The successful mapping of ~90 % of the SCAR markers to the ASGR in the Pennisetum F₁ mapping population shows that DSN normalization and Illumina sequencing can be used as an effective strategy for targeted mapping of a physically large locus rich in repetitive sequences, like that of the ASGR.     

The Superagonistic Activity of Bovine Thyroid-stimulating Hormone (TSH) and the Human TR1401 TSH Analog Is Determined by Specific Amino Acids in the Hinge Region of the Human TSH Receptor

Bovine TSH (bTSH) has a higher affinity to the human TSHR (hTSHR) and a higher signaling activity than human TSH (hTSH). The molecular reasons for these phenomena are unknown. Distinct negatively charged residues (Glu²⁹⁷, Glu³⁰³, and Asp³⁸²) in the hinge region of the hTSHR are known to be important for bTSH binding and signaling. To investigate the potential relevance of these positions for differences between bTSH and hTSH in the interaction to the hTSHR, we determined bTSH- and hTSH-mediated cAMP production of several substitutions at these three hinge residues. To examine specific variations of hTSH, we also investigated the superagonistic hTSH analog TR1401 (TR1401), whose sequence differs from hTSH by four additional positively charged amino acids that are also present in bTSH. To characterize possible interactions between the acidic hTSHR positions Glu²⁹⁷, Glu³⁰³, or Asp³⁸² and the additional basic residues of TR1401, we investigated TR1401 binding and signaling properties. Our data reveal increased cAMP signaling of the hTSHR using TR1401 and bTSH compared with hTSH. Whereas Asp³⁸² seems to be important for bTSH- and TR1401-mediated but not for hTSH-mediated signaling, the substitution E297K exhibits a decreased signaling for all three TSH variants. Interestingly, bTSH and TR1401 showed only a slightly different binding pattern. These observations imply that specific residues of the hinge region are mediators of the superagonistic activity of bTSH and TR1401 in contrast to hTSH. Moreover, the simultaneous localization of binding components in the glycoprotein hormone molecule and the receptor hinge region permits important reevaluation of interacting hormone receptor domains.It is well known that bovine TSH (bTSH)² has a higher affinity to the human TSHR (hTSHR) and a 6–10-fold higher intrinsic signaling activity than human TSH (hTSH) (1–5). Human TSH and bTSH share high amino acid sequence identity in the α-subunit (74.1%) and β-subunit (88.4%) (6). Studies involving fusion of hTSH and bTSH α- and β-subunits indicate that the higher affinity and the superagonistic cAMP activity of bTSH at the hTSHR depend primarily on amino acid sequences of the bTSH α-subunit (6). The most noticeable sequence differences between bovine and human TSH consist of four positively charged residues located in the surface-exposed loops of the α-subunit and one positively charged residue in the β-subunit of bTSH (Fig. 1). Moreover, it has previously been shown that positively charged α loop 1 (α-L1) residues are important for the high bioactivity of bTSH, and they have been implicated in receptor binding. These specific characteristics led to the generation of superagonistic hTSH analogs (6). The human TSH analog TR1401 and bTSH differ from hTSH most importantly by four additional positively charged amino acids located in close spatial proximity at the α-L1, of which three are located at identical positions in bTSH and TR1401 (Fig. 1).Open in a separate windowFIGURE 1.Sequence differences between TSH variants used in the present study. A, alignment of the α- and β-subunit of the hTSH (SwissProt: GLHA_HUMAN {"type":"entrez-protein","attrs":{"text":"P01215","term_id":"121312","term_text":"P01215"}}P01215, TSHB_HUMAN {"type":"entrez-protein","attrs":{"text":"P01222","term_id":"311033515","term_text":"P01222"}}P01222), bTSH (GLHA_BOVIN {"type":"entrez-protein","attrs":{"text":"P01217","term_id":"121310","term_text":"P01217"}}P01217, TSHB_BOVIN {"type":"entrez-protein","attrs":{"text":"P01223","term_id":"136442","term_text":"P01223"}}P01223), and the superagonistic hTSH analog TR1401. The additional positively charged residues at TR1401 and at bTSH compared with wt hTSH are boxed in blue. Sequence numbering for human TSH and human analog TR1401 without signal peptide is shown in blue. B, three-dimensional structural TSH models illustrating the spatial localization of the charge related sequence differences between the TSH variants. The TSH α-subunit is shown in gray, and the β-subunit is in orange. Positively charged residues are highlighted in blue, and the C-α atoms of additional positively charged residues compared with hTSH are highlighted by blue globes. Panel i, bovine TSH, characterized by four additional positively charged residues in the α-L1 (T11K, Q13K, P16K, and Q20K) and one positively charged residue in the β-L3 (L69R); panel ii, human TSH without positively charged residues in the α-L1 and β-L3; and panel iii, the human TSH analog TR1401 is characterized by four additional positively charged residues in the α-L1 (Q13K, E14K, P16K, and Q20K) but shows a lack of the additional positively charged residue in the β-L3.TSH binds to the large extracellular region of its receptor. The extracellular region of the TSHR consists of the leucine-rich repeat domain (LRRD), which is linked with the membrane-spanning serpentine domain by the hinge region. Recently, the binding arrangements between the homologous FSH and a part of the FSH receptor ectodomain including the LRRD (FSH receptor amino acids Cys¹⁸–Ala²⁴⁶) have been identified (7). However, the hinge region is not contained in this x-ray structure (7).In vitro data provide convincing evidence for the functional importance of the hinge region for receptor activation and TSH binding (8–22). Recently, we specified positions Glu²⁹⁷ and Glu³⁰³ in the N-terminal portion and Asp³⁸² in the C-terminal portion of the hTSHR hinge region as important for bTSH binding, suggesting that in the process of bTSH binding an extended hormone-binding site is obviously essential (18). The negative charge of positions Glu²⁹⁷ and Asp³⁸² likely interact with positively charged residues of bTSH by complementary charge-charge interaction (18).To elucidate whether these hinge residues of the hTSHR are specific for interaction with bTSH, we investigated the functional characteristics of the hTSH analog TR1401 and the native ligand hTSH. For the comparison of these two TSH variants with bTSH, we used several mutations and alanine combinations at the signaling and bTSH binding-sensitive hTSHR hinge positions Glu²⁹⁷, Glu³⁰³, and Asp³⁸². Our data indicate that the higher bioactivity of the TSH variants TR1401 and bTSH are mediated by specific charged residues of the hormone and the hinge region of the hTSHR. Our findings also support the concept that the hinge region of the TSHR is an modulator of TSH potency and efficacy.