GDF15 linked to maternal risk of nausea and vomiting during pregnancy.

GDF15, a hormone acting on the brainstem, has been implicated in the nausea and vomiting of pregnancy, including its most severe form, hyperemesis gravidarum (HG), but a full mechanistic understanding is lacking1-4. Here we report that fetal production of GDF15 and maternal sensitivity to it both contribute substantially to the risk of HG. We confirmed that higher GDF15 levels in maternal blood are associated with vomiting in pregnancy and HG. Using mass spectrometry to detect a naturally labelled GDF15 variant, we demonstrate that the vast majority of GDF15 in the maternal plasma is derived from the feto-placental unit. By studying carriers of rare and common genetic variants, we found that low levels of GDF15 in the non-pregnant state increase the risk of developing HG. Conversely, women with ß-thalassaemia, a condition in which GDF15 levels are chronically high5, report very low levels of nausea and vomiting of pregnancy. In mice, the acute food intake response to a bolus of GDF15 is influenced bi-directionally by prior levels of circulating GDF15 in a manner suggesting that this system is susceptible to desensitization. Our findings support a putative causal role for fetally derived GDF15 in the nausea and vomiting of human pregnancy, with maternal sensitivity, at least partly determined by prepregnancy exposure to the hormone, being a major influence on its severity. They also suggest mechanism-based approaches to the treatment and prevention of HG.

KRAS mutational subtype and copy number predict in vitro response of human pancreatic cancer cell lines to MEK inhibition.

BACKGROUND: To study the molecular mechanism regulating sensitivity to MEK inhibition in pancreatic cancer cell lines. METHODS: A growth inhibition assay determined sensitivity to MEK162 in a panel of 29 pancreatic cancer cell lines. For the same panel, KRAS mutational status and copy-number variation (CNV) was determine using PCR, array CGH and FISH. Two sensitive and two resistant cell lines were further interrogated for difference in baseline and MEK162-induced gene expression, as well as signal transduction using microarray and western blotting. Cell cycle and apoptosis analysis was measured by flow cytometry. RESULTS: We report a strong correlation between both specific KRAS mutational subtype and CNV, and sensitivity to MEK inhibition. Cell lines with a KRAS (V12) mutation and KRAS gains or loss (n=7) are ∼10 times more resistant than those having neither a KRAS (V12) mutation nor KRAS CNV (n=14). Significant differences in baseline and MEK162-induced gene expression exist between the sensitive and resistant lines, especially in genes involved in RAS, EGF receptor and PI3K pathways. This was further supported by difference in signal transduction. MEK 162 blocked ERK1/2, as well as inhibited PI3K and S6 and increased p27KIP1 levels in the sensitive lines. CONCLUSIONS: Given the potency of MEK162, it may be a promising new therapy for patients with pancreatic cancer and KRAS mutational subtypes, and CNV may serve as important biomarkers for selecting patients that benefit from MEK-targeting based on these preclinical data.

Fetally-encoded GDF15 and maternal GDF15 sensitivity are major determinants of nausea and vomiting in human pregnancy.

Human pregnancy is frequently accompanied by nausea and vomiting that may become severe and life-threatening, as in hyperemesis gravidarum (HG), the cause of which is unknown. Growth Differentiation Factor-15 (GDF15), a hormone known to act on the hindbrain to cause emesis, is highly expressed in the placenta and its levels in maternal blood rise rapidly in pregnancy. Variants in the maternal GDF15 gene are associated with HG. Here we report that fetal production of GDF15, and maternal sensitivity to it, both contribute substantially to the risk of HG. We found that the great majority of GDF15 in maternal circulation is derived from the feto-placental unit and that higher GDF15 levels in maternal blood are associated with vomiting and are further elevated in patients with HG. Conversely, we found that lower levels of GDF15 in the non-pregnant state predispose women to HG. A rare C211G variant in GDF15 which strongly predisposes mothers to HG, particularly when the fetus is wild-type, was found to markedly impair cellular secretion of GDF15 and associate with low circulating levels of GDF15 in the non-pregnant state. Consistent with this, two common GDF15 haplotypes which predispose to HG were associated with lower circulating levels outside pregnancy. The administration of a long-acting form of GDF15 to wild-type mice markedly reduced subsequent responses to an acute dose, establishing that desensitisation is a feature of this system. GDF15 levels are known to be highly and chronically elevated in patients with beta thalassemia. In women with this disorder, reports of symptoms of nausea or vomiting in pregnancy were strikingly diminished. Our findings support a causal role for fetal derived GDF15 in the nausea and vomiting of human pregnancy, with maternal sensitivity, at least partly determined by pre-pregnancy exposure to GDF15, being a major influence on its severity. They also suggest mechanism-based approaches to the treatment and prevention of HG.

The psychosocial burden of hyperemesis gravidarum.

OBJECTIVE: To describe the psychosocial burden of hyperemesis gravidarum (HG) in a large cohort of affected women, focusing on previously unreported problems. STUDY DESIGN: Women with HG described their pregnancy history in an open-ended survey administered internationally through an HG website during 2003 to 2005. RESULT: Of the 808 participants, 626 (77.5%) were American. A large majority (82.8%) reported that HG caused negative psychosocial changes, consisting of (1) socioeconomic changes, for example, job loss or difficulties, (2) attitude changes including fear regarding future pregnancies and (3) psychiatric sequelae, for example, feelings of depression and anxiety, which for some continued postpartum. Women who reported that their health-care provider was uncaring or unaware of the severity of their symptoms were nearly twice as likely to report these psychiatric sequelae (odds ratio 1.86, 95% confidence interval 1.06 to 3.29, P=0.032). CONCLUSION: Over 80% of a large cohort of women with HG reported that HG caused a negative psychosocial impact.

No increased risk of psychological/behavioral disorders in siblings of women with hyperemesis gravidarum (HG) unless their mother had HG.

Hyperemesis gravidarum (HG), severe nausea and vomiting of pregnancy, is characterized by prolonged maternal stress, undernutrition and dehydration. Maternal stress and malnutrition of pregnancy are linked to poor neonatal outcome and associated with poor adult health, and we recently showed that in utero exposure to HG may lead to increased risks of psychological and behavioral disorders in the offspring. In addition, we have shown familial aggregation of HG, which is strong evidence for a genetic component to the disease. In this study, we compare the rates of psychological and behavioral disorders in 172 adults with and 101 adults without a sibling with HG. The rate of emotional/behavioral disorders is identical (15%) in both groups. The results suggest that the etiology of HG is not likely to include genetic factors associated with emotional and behavioral disorders. In addition, this study provides evidence that the increased incidence of psychological/behavioral disorders among offspring of women with HG is attributable to the HG pregnancy itself, rather than to confounding genetic factors linked to HG.

Prenatal exposure to hyperemesis gravidarum linked to increased risk of psychological and behavioral disorders in adulthood.

Hyperemesis gravidarum (HG), severe nausea and vomiting of pregnancy, is characterized by long-term maternal stress, undernutrition and dehydration. While maternal stress and malnutrition of pregnancy are linked to poor neonatal outcome and associated with poor adult health, long-term outcome of fetal exposure to HG has never been explored. The purpose of this study is to determine whether long-term emotional and behavioral diagnoses may be associated with fetal exposure to HG. Emotional and behavioral diagnoses of adults born of a pregnancy complicated by HG were compared to diagnoses from non-exposed controls. Offspring exposed to HG in utero were significantly more likely to have a psychological and behavioral disorder (OR = 3.6, P < 0.0001) with diagnoses primarily of depression, bipolar disorder and anxiety. In utero exposure to HG may lead to increased risks of psychological and behavioral disorders in the offspring.

Activity of lapatinib a novel HER2 and EGFR dual kinase inhibitor in human endometrial cancer cells.

In this study, we explore the therapeutic potential of lapatinib a selective inhibitor of both the EGFR and HER2 tyrosine kinases for the treatment of endometrial cancer. The effect of lapatinib on tumour cell growth and receptor activation was studied in a panel of human endometrial cancer cell lines. Candidate molecular markers predicting sensitivity were assessed by baseline gene expression profiling, ELISA, and western blot analyses. Multiple drug effect/combination index (CI) isobologram analysis was used to study the interactions between chemotherapeutic drugs and lapatinib. Concentration-dependent anti-proliferative effects of lapatinib were seen in all endometrial cancer cell lines tested, but varied significantly between individual cell lines (IC(50) range: 0.052-10.9 micromol). HER2 overexpression or increased expression of EGFR was significantly associated with in vitro sensitivity (P=0.024 or 0.011, respectively). Lapatinib exerts growth inhibition in a PTEN-independent manner. Sensitive cell lines also exhibited increased expression of EGFR ligands or HER3. In contrast, lapatinib-resistant cell lines exhibited high androgen receptor (AR) levels or epithelial-to-mesenchymal transition (post-EMT) features. In endometrial cancer cells, at a wide range of clinically achievable drug concentrations, additive and synergistic interactions were observed for lapatinib plus carboplatin, paclitaxel, docetaxel, and doxorubicin. These observations provide a clear biologic rational to test lapatinib as a single agent or in combination with chemotherapy in endometrial cancer with HER2 overexpression. Expression of EGFR, its ligands, HER3, AR, and post-EMT markers warrant further evaluation to help define patients with HER2-nonoverexpressing endometrial cancer most likely to benefit from lapatinib.

Frozen tumor tissue microarray technology for analysis of tumor RNA, DNA, and proteins.

Tissue microarray technology is a new method used to analyze several hundred tumor samples on a single slide allowing high throughput analysis of genes and proteins on a large cohort. The original methodology involves coring tissues from paraffin-embedded tissue donor blocks and placing them into a single paraffin block. One difficulty with paraffin-embedded tissue relates to antigenic changes in proteins and mRNA degradation induced by the fixation and embedding process. We have modified this technology by using frozen tissues embedded in OCT compound as donor samples and arraying the specimens into a recipient OCT block. Tumor tissue is not fixed before embedding, and sections from the array are evaluated without fixation or postfixed according to the appropriate methodology used to analyze a specific gene at the DNA, RNA, and/or protein levels. While paraffin tissue arrays can be problematic for immunohistochemistry and for RNA in situ hybridization analyses, this method allows optimal evaluation by each technique and uniform fixation across the array panel. We show OCT arrays work well for DNA, RNA, and protein analyses, and may have significant advantages over the original technology for the assessment of some genes and proteins by improving both qualitative and quantitative results.

The ompA 5' untranslated region impedes a major pathway for mRNA degradation in Escherichia coli.

The unusual longevity of the Escherichia coli ompA transcript is determined by its 5' untranslated region (UTR), which functions in vivo as an mRNA stabilizer. Here we show that this 5' UTR can prolong the lifetime in E. coli of a variety of heterologous mRNAs to which it is joined, either as a gene fusion or as an operon fusion. Statistical extrapolation suggests that it is quite likely that most E. coli mRNAs could be stabilized in this manner. We conclude that the ompA 5' UTR impedes a major pathway for mRNA degradation in E. coli and that stabilization by fusion to this UTR does not require translational readthrough of the heterologous mRNA segment by ribosomes that initiate translation at the ompA ribosome-binding site. Additional experiments indicate that the E. coli ribonuclease whose action is slowed by the ompA 5' UTR is not RNase III.

Molecular cytogenetic analysis of consistent abnormalities at 8q12-q22 in breast cancer.

Studies using comparative genomic hybridization (CGH) indicate that portions of chromosome arm 8q from 8q12 to 8qter are present at an increased relative copy number in a broad range of solid tumors. In this study we define an approximately 1 Mb wide region that appears to be frequently abnormal in copy number or structure in breast cancer cell lines and primary tumors. This was accomplished by fluorescence in situ hybridization (FISH) with yeast artificial chromosomes (YACs) mapped to 8q2-q22. Eleven breast cancer cell lines and ten primary tumors were analyzed. A minimal region of rearrangement was localized to the CEPH-YAC 928F9 in three breast cancer cell lines with unbalanced translocation breakpoints mapping in this region. Unbalanced translocations also were detected in two primary tumors mapping between CEPH-YAC clones 890C4 and 936B3, flanking 928F9. An increased copy number in the minimal region was detected in nine cell lines and in multiple primary tumors. This suggests the possibility that a single gene mapping to 928F9 is involved in breast cancer development or progression and may be deregulated by copy number increases in some tumors and by translocation in others. Four expressed sequence tags were mapped to YAC 928F9 and analyzed for rearrangements by Southern analysis and for abnormal expression by Northern analysis.

Characterization of genes encoding known and novel human mast cell tryptases on chromosome 16p13.3.

Tryptases are serine proteases implicated in asthma and are very highly expressed in human mast cells. They fall into two groups, alpha and beta. Although several related tryptase mRNAs are known, it is unclear which if any are transcripts of separate haploid genes. The studies described here investigated the nature and number of human tryptases and sought possibly novel members of the family. To this end, two human bacterial artificial chromosome (BAC) clones containing tryptase genes were identified and mapped to chromosome 16p13.3, of which approximately 2.2 megabases are syntenic with the part of mouse chromosome 17 containing tryptase genes mouse mast cell protease (mMCP)-6 and -7. Sequencing and restriction mapping suggest that the BACs may partially overlap. Sequenced BAC genes correspond to three known beta-tryptases (betaI, betaII, and betaIII), an alpha-like gene, and a pair of novel hybrid genes related partly to alpha/beta-tryptases and partly to orthologs of mMCP-7. betaII and betaIII, betaI and alphaII, as well as the two mMCP-7-like genes, may be alleles at single loci; in total, there are at least three nonallelic tryptase genes in the isolated BAC clones. DNA blotting and restriction analysis suggest that the BACs include most members of the immediate tryptase family. Thus, chromosome 16p13.3 harbors a cluster of known and previously undescribed members of the tryptase gene family.

The hD52 (TPD52) gene is a candidate target gene for events resulting in increased 8q21 copy number in human breast carcinoma.

Chromosome band 8q21 is frequently overrepresented in human cancer, but to date no 8q21 target gene has been proposed. The hD52 (TPD52) gene is of potential significance in breast and other cancers due to its location and expression pattern. Fine mapping of hD52 placed this locus within the peak of the 8q21 amplicon delineated in the SK-BR-3 breast carcinoma cell line, and a positive association between hD52 gene dosage and transcript levels was subsequently demonstrated in four breast carcinoma cell lines, including SK-BR-3. Increased copy number (ICN) was measured using Southern blot analyses in 3/32 human breast carcinomas at hD52, and the related hD54 gene in 20q13.2-q13.3. Subsequent immunohistochemical analysis of hD52 expression in 19 breast carcinomas with varying hD52 gene dosages demonstrated a significant positive association between hD52 dosage and hD52 expression using a Spearman rank correlation coefficient (r(s) = 0.573, alpha = 0.01) and a Wilcoxon rank-sum test (alpha = 0.05). On the basis of its map location and expression pattern in breast carcinoma, we therefore propose hD52 as a candidate target gene at chromosome band 8q21.

Disruption of the architectural factor HMGI-C: DNA-binding AT hook motifs fused in lipomas to distinct transcriptional regulatory domains.

Lipomas are one of the most common mesenchymal neoplasms in humans. They are characterized by consistent cytogenetic aberrations involving chromosome 12 in bands q14-15. Interestingly, this region is also the site of rearrangement for other mesenchymally derived tumors. This study demonstrates that HMGI-C, an architectural factor that functions in transcriptional regulation, has been disrupted by rearrangement at the 12q14-15 chromosomal breakpoint in lipomas. Chimeric transcripts were isolated from two lipomas in which HMGI-C DNA-binding domains (AT hook motifs) are fused to either a LIM or an acidic transactivation domain. These results, identifying a gene rearranged in a benign neoplastic process that does not proceed to a malignancy, suggest a role for HMGI-C in adipogenesis and mesenchyme differentiation.

Analysis of androgen receptor DNA reveals the independent clonal origins of uterine leiomyomata and the secondary nature of cytogenetic aberrations in the development of leiomyomata.

Uterine leiomyomata are thought to be monoclonal neoplasms. Accordingly, investigations of clonality with G6PD isoforms used as a marker for X chromosome inactivation have suggested independent origins for multiple tumors within individual uteri. However, results from a recent study assessing methylation differences between DNA of active and inactive X chromosomes have been interpreted to suggest that multiple tumors may arise from a common precursor. We have examined the clonality of 36 leiomyomata from 16 patients by analyzing X chromosome inactivation as indicated by the methylation status of the X-linked androgen receptor gene. As shown by this assay, all informative leiomyomata were monoclonal in origin. In patients with multiple leiomyomata, a random distribution of inactivation between the X homologs was noted, consistent with an independent origin of each tumor. Cytogenetic analysis was also performed on short-term cell cultures of 27 of the 36 tumors. In each of two tumors that had both cells with a clonal karyotypic abnormality and karyotypically normal cells, DNA prepared from short-term cultures showed a monoclonal pattern of X inactivation identical to that of the leiomyoma from which they were derived. These data suggest that karyotypically normal cells present in short-term cultures of uterine leiomyomata are part of the tumor clone, and that clonal expansion of tumor cells precedes the development of cytogenetic aberrations.

Translocation breakpoints upstream of the HMGIC gene in uterine leiomyomata suggest dysregulation of this gene by a mechanism different from that in lipomas.

Uterine leiomyomata are the most common pelvic tumors in women and are the indication for more than 200,000 hysterectomies annually in the United States. Rearrangement of chromosome 12 in bands q14-q15 is characteristic of uterine leiomyomata and other benign mesenchymal tumors, and we identified a yeast artificial chromosome (YAC) spanning chromosome 12 translocation breakpoints in a uterine leiomyoma, a pulmonary chondroid hamartoma, and a lipoma. Recently, we demonstrated that HMGIC, which is an architectural factor mapping within the YAC, is disrupted in lipomas, resulting in novel fusion transcripts. Here, we report on the localization of translocation breakpoints in seven uterine leiomyomata from 10 to > 100 kb upstream of HMGIC by use of fluorescence in situ hybridization. Our findings suggest a different pathobiologic mechanism in uterine leiomyomata from that in lipomas. HMGIC is the first gene identified in chromosomal rearrangements in uterine leiomyomata and has important implications for an understanding of benign mesenchymal proliferation and differentiation.

Identification of a YAC spanning the translocation breakpoints in uterine leiomyomata, pulmonary chondroid hamartoma, and lipoma: physical mapping of the 12q14-q15 breakpoint region in uterine leiomyomata.

Uterine leiomyomata are the most common tumors in women and can cause abnormal uterine bleeding, pelvic pain, and infertility. Approximately 200,000 hysterectomies are performed annually in the U.S. to relieve patients of the medical sequelae of these benign neoplasms. Our efforts have focused on cloning the t(12;14)(q14-q15;q23-q24) breakpoint in uterine leiomyoma to further our understanding of the biology of these tumors. Thirty-nine YACs and six cosmids mapping to 12q14-q15 have been mapped by fluorescence in situ hybridization to tumor metaphase chromosomes containing a t(12;14). One YAC spanned the translocation breakpoint and was mapped to tumor metaphases from a pulmonary chondroid hamartoma containing a t(12;14)(q14-q15;q23-q24) and a lipoma containing a t(12;15)(q15;q24); this YAC also spanned the breakpoint in these two tumors, suggesting that the same gene on chromosome 12 may be involved in the pathobiology of these distinct benign neoplasms.

Identification, characterization, and chromosomal localization of the human homolog (hES) of ES/130.

The chicken extracellular matrix glycoprotein ES/ 130 is necessary for epithelial--mesenchymal transformation in the developing heart and is also expressed in noncardiac chicken tissues such as limb and notochord. We have identified hES, the human homolog of chicken ES/130. Fluorescence in situ hybridization analysis (FISH) localizes hES to human chromosome 20p11.2-p12. FISH analyses of individuals with 20p12 deletions and affected by Alagille syndrome exclude hES as a candidate gene for this disorder. Reverse transcriptase-polymerase chain reaction studies reveal that hES is expressed in both fetal and adult human tissues and that hES expression in the left ventricle is increased in the failing adult heart. Further studies will evaluate how hES mutations may relate to congenital human cardiac and skeletal anomalies as well as cardiac remodeling in the adult.