On the basis of developmental gene expression, the vertebrate central nervous system comprises: a forebrain plus anterior midbrain, a midbrain-hindbrain boundary region (MHB) having organizer properties, and a rhombospinal domain. The vertebrate MHB is characterized by position, by organizer properties and by being the early site of action of Wnt1 and engrailed genes, and of genes of the Pax2/5/8 subfamily. Wada and others (Wada, H., Saiga, H., Satoh, N. and Holland, P. W. H. (1998) Development 125, 1113-1122) suggested that ascidian tunicates have a vertebrate-like MHB on the basis of ascidian Pax258 expression there. In another invertebrate chordate, amphioxus, comparable gene expression evidence for a vertebrate-like MHB is lacking. We, therefore, isolated and characterized AmphiPax2/5/8, the sole member of this subfamily in amphioxus. AmphiPax2/5/8 is initially expressed well back in the rhombospinal domain and not where a MHB would be expected. In contrast, most of the other expression domains of AmphiPax2/5/8 correspond to expression domains of vertebrate Pax2, Pax5 and Pax8 in structures that are probably homologous - support cells of the eye, nephridium, thyroid-like structures and pharyngeal gill slits; although AmphiPax2/5/8 is not transcribed in any structures that could be interpreted as homologues of vertebrate otic placodes or otic vesicles. In sum, the developmental expression of AmphiPax2/5/8 indicates that the amphioxus central nervous system lacks a MHB resembling the vertebrate isthmic region. Additional gene expression data for the developing ascidian and amphioxus nervous systems would help determine whether a MHB is a basal chordate character secondarily lost in amphioxus. The alternative is that the MHB is a vertebrate innovation.

Using voltage and pH reporter dyes, we have discovered a never-before-seen regionalization of the Xenopus ectoderm, with cell subpopulations delimited by different membrane voltage and pH. We distinguished three courses of bioelectrical activity. Course I is a wave of hyperpolarization that travels across the gastrula. Course II comprises the appearance of patterns that match shape changes and gene expression domains of the developing face; hyperpolarization marks folding epithelium and both hyperpolarized and depolarized regions overlap domains of head patterning genes. In Course III, localized regions of hyperpolarization form at various positions, expand, and disappear. Inhibiting H(+) -transport by the H(+) -V-ATPase causes abnormalities in: (1) the morphology of craniofacial structures; (2) Course II voltage patterns; and (3) patterns of sox9, pax8, slug, mitf, xfz3, otx2, and pax6. We conclude that this bioelectric signal has a role in development of the face. Thus, it exemplifies an important, under-studied mechanism of developmental regulation.

The reproductive role of the fallopian tube is to transport the sperm and egg. The tube is positioned to act as a bridge between the ovary where the egg is released and the uterus where implantation occurs. Throughout reproductive years, the fallopian tube epithelium undergoes repetitive damage and regeneration. Although a reservoir of adult epithelial stem cells must exist to replenish damaged cells, they remain unidentified. Here, we report isolation of a subset of basally located human fallopian tube epithelia (FTE) that lack markers of ciliated (β-tubulin; TUBB4) or secretory (PAX8) differentiated cells. These undifferentiated cells expressed cell surface antigens: epithelial cell adhesion molecule, CD44, and integrin α 6. This FTE subpopulation was fivefold enriched for cells capable of clonal growth and self-renewal suggesting that they contain the FTE stem-like cells (FTESCs). A twofold enrichment of the FTESC was found in the distal compared to the proximal end of the tube. The distal fimbriated end of the fallopian tube is a well-characterized locus for initiation of serous carcinomas. An expansion of the cells expressing markers of FTESC was detected in tubal intraepithelial carcinomas and in fallopian tubes from patients with invasive serous cancer. These findings suggest that FTESC may play a role in the initiation of serous tumors. Characterization of these stem-like cells will provide new insight into how the FTE regenerate, respond to injury, and may initiate cancer.

Thyroid hormone (TH) is essential for the development of hearing. Lack of TH in a critical developmental period from embryonic day 17 to postnatal day 12 (P12) in rats and mice leads to morphological and functional deficits in the organ of Corti and the auditory pathway. We investigated the effects of TH on inner hair cells (IHCs) using patch-clamp recordings, capacitance measurements, and immunocytochemistry in hypothyroid rats and athyroid Pax8-/- mice. Spontaneous and evoked Ca2+ action potentials (APs) were present in control IHCs from P3-P11 rats and vanished in parallel with the expression of a rapidly activating Ca2+- and voltage-activated K+ (BK) conductance. IHCs of hypothyroid rats and athyroid Pax8-/- mice displayed APs until the end of the third postnatal week because of threefold elevated Ca2+ currents and missing expression of BK currents. After the fourth postnatal week, some IHCs showed BK currents whereas adjacent IHCs did not, demonstrated by electrophysiology and immunocytochemistry. To test whether the prolonged spiking activity during TH deficiency may be transmitted at IHC synapses, capacitance measurements were performed in parallel to analysis of otoferlin expression, a protein thought to play an essential role in exocytosis of IHCs. Strikingly, otoferlin was absent from IHCs of hypothyroid rats but not of Pax8-/- mice, although both cell types showed exocytosis with an efficiency typical for immature IHCs. These results demonstrate for the first time a TH-dependent control of IHC spiking activity before the onset of hearing attributable to effects of TH on Ca2+ and BK channels. Moreover, they question an indispensable role of otoferlin for exocytosis in IHCs.

The role of vascular endothelial growth factor (VEGF) in renal fibrosis, tubular cyst formation, and glomerular diseases is incompletely understood. We studied a new conditional transgenic mouse system [Pax8-rtTA/(tetO)(7)VEGF], which allows increased tubular VEGF production in adult mice. The following pathology was observed. The interstitial changes consisted of a ubiquitous proliferation of peritubular capillaries and fibroblasts, followed by deposition of matrix leading to a unique kind of fibrosis, ie, healthy tubules amid a capillary-rich dense fibrotic tissue. In tubular segments with high expression of VEGF, cysts developed that were surrounded by a dense network of peritubular capillaries. The glomerular effects consisted of a proliferative enlargement of glomerular capillaries, followed by mesangial proliferation. This resulted in enlarged glomeruli with loss of the characteristic lobular structure. Capillaries became randomly embedded into mesangial nodules, losing their filtration surface. Serum VEGF levels were increased, whereas endogenous VEGF production by podocytes was down-regulated. Taken together, this study shows that systemic VEGF interferes with the intraglomerular cross-talk between podocytes and the endocapillary compartment. It suppresses VEGF secretion by podocytes but cannot compensate for the deficit. VEGF from podocytes induces a directional effect, attracting the capillaries to the lobular surface, a relevant mechanism to optimize filtration surface. Systemic VEGF lacks this effect, leading to severe deterioration in glomerular architecture, similar to that seen in diabetic nephropathy.

Recently, a translocation t(2; 3)(q13;p25), involving the fusion of PAX8 and peroxisome proliferator-activated receptor gamma (PPAR gamma) was suggested to arise only in follicular thyroid carcinomas. In this study, a group of 87 thyroid tumors were analyzed to determine the involvement of the PAX8/PPAR gamma fusion gene in these tumors, and also to determine whether this rearrangement can be used as a diagnostic marker for the differentiation between follicular thyroid carcinoma and adenoma. The PAX8/PPAR gamma rearrangement was detected by RT-PCR, fluorescence in situ hybridization, and/or Western analysis in 10 of 34 (29%) follicular thyroid carcinomas and in one of 20 (5%) atypical follicular thyroid adenomas, but not in any of the 20 follicular thyroid adenomas or 13 anaplastic thyroid carcinomas studied. In addition, seven of the 87 thyroid tumors exhibited involvement of PPAR gamma alone. Our findings suggest that PAX8/PPAR gamma occurs frequently in follicular thyroid carcinomas, and the presence of this rearrangement is likely to prove highly suggestive of a malignant tumor. Lack of the PAX8/PPAR gamma rearrangement in the anaplastic thyroid carcinoma group suggests that the tumorigenic pathway in these tumors is likely to be independent of this fusion. Furthermore, the results suggest that other rearrangements, involving PPAR gamma and other unidentified genes, may be involved in follicular thyroid tumorigenesis.

Pelvic serous cancer is a diverse disease, and the assignment of primary site -- ovarian, tubal, or peritoneal -- is often problematic. Recent studies indicate that a proportion of these tumors arise from the distal fallopian tube, originating as serous tubal intraepithelial carcinoma (STIC). This study examined the relationship of 2 parameters for assigning origin -- endosalpingeal involvement and dominant ovarian mass -- in the context of STIC. Endometrioid carcinomas served as a reference. Eighty-seven consecutive pelvic serous cancers in which the tubes and ovaries were completely examined (SEE-FIM protocol) were analyzed. The presence of a dominant ovarian mass (DOM+), involvement of the fimbrial mucosa (FIM+), and STIC were correlated. In addition, tumor categories were compared with respect to PAX8, p73, p53, and p16 immunohistochemistry. Of the 27 DOM+ cases, 13 (48%) were FIM+ and a STIC was present in 3 (11%). Of the 60 DOM(-) cases, 48 (78%) were FIM+ and 28 (45%) harbored a STIC. In 92% of all cases, tumor distribution was extensive with bilateral ovarian and extraovarian peritoneal involvement. All tumor categories were immunophenotypically similar. In contrast, DOM+, FIM+, and STIC were found in 81%, 19%, and 0% of ovarian endometrioid carcinomas. In conclusion, there is a significant inverse relationship between DOM+ and STIC (P=0.001), indicating both parameters are of value in grouping pelvic serous carcinomas more likely to be ovarian [DOM+/FIM(-)] versus fimbrial [DOM(-)/STIC], and ovarian or peritoneal surface (DOM-/FIM-) in origin. Nevertheless, the shared immunophenotype suggests a common cell of origin for all categories, irrespective of site.

Ribbon synapses of inner hair cells (IHCs) undergo developmental maturation until after the onset of hearing. Here, we studied whether IHC synaptogenesis is regulated by thyroid hormone (TH). We performed perforated patch-clamp recordings of Ca2+ currents and exocytic membrane capacitance changes in IHCs of athyroid and TH-substituted Pax8-/- mice during postnatal development. Ca2+ currents remained elevated in athyroid IHCs at the end of the second postnatal week, when it had developmentally declined in wild-type and TH-rescued mutant IHCs. The efficiency of Ca2+ influx in triggering exocytosis of the readily releasable vesicle pool was reduced in athyroid IHCs. Ribbon synapses were formed despite the TH deficiency. However, different from wild type, in which synapse elimination takes place at approximately the onset of hearing, the number of ribbon synapses remained elevated in 2-week-old athyroid IHCs. Moreover, the ultrastructure of these synapses appeared immature. Using quantitative reverse transcription-PCR, we found a TH-dependent developmental upregulation of the mRNAs for the neuronal SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins, SNAP25 (synaptosomal-associated protein of 25 kDa) and synaptobrevin 1, in the organ of Corti. These molecular changes probably contribute to the improvement of exocytosis efficiency in mature IHCs. IHCs of 2-week-old athyroid Pax8-/- mice maintained the normally temporary efferent innervation. Moreover, they lacked large-conductance Ca2+-activated K+ channels and KCNQ4 channels. This together with the persistently increased Ca2+ influx permitted continued action potential generation. We conclude that TH regulates IHC differentiation and is essential for morphological and functional maturation of their ribbon synapses. We suggest that presynaptic dysfunction of IHCs is a mechanism in congenital hypothyroid deafness.

Disrupted circadian rhythms and reduced sleep duration are associated with several human diseases, particularly obesity and type 2 diabetes, but until recently, little was known about the genetic factors influencing these heritable traits. We performed genome-wide association studies of self-reported chronotype (morning/evening person) and self-reported sleep duration in 128,266 white British individuals from the UK Biobank study. Sixteen variants were associated with chronotype (P<5x10-8), including variants near the known circadian rhythm genes RGS16 (1.21 odds of morningness, 95% CI [1.15, 1.27], P = 3x10-12) and PER2 (1.09 odds of morningness, 95% CI [1.06, 1.12], P = 4x10-10). The PER2 signal has previously been associated with iris function. We sought replication using self-reported data from 89,283 23andMe participants; thirteen of the chronotype signals remained associated at P<5x10-8 on meta-analysis and eleven of these reached P<0.05 in the same direction in the 23andMe study. We also replicated 9 additional variants identified when the 23andMe study was used as a discovery GWAS of chronotype (all P<0.05 and meta-analysis P<5x10-8). For sleep duration, we replicated one known signal in PAX8 (2.6 minutes per allele, 95% CI [1.9, 3.2], P = 5.7x10-16) and identified and replicated two novel associations at VRK2 (2.0 minutes per allele, 95% CI [1.3, 2.7], P = 1.2x10-9; and 1.6 minutes per allele, 95% CI [1.1, 2.2], P = 7.6x10-9). Although we found genetic correlation between chronotype and BMI (rG = 0.056, P = 0.05); undersleeping and BMI (rG = 0.147, P = 1x10-5) and oversleeping and BMI (rG = 0.097, P = 0.04), Mendelian Randomisation analyses, with limited power, provided no consistent evidence of causal associations between BMI or type 2 diabetes and chronotype or sleep duration. Our study brings the total number of loci associated with chronotype to 22 and with sleep duration to three, and provides new insights into the biology of sleep and circadian rhythms in humans.

Ovarian low-grade serous carcinomas are thought to evolve in a stepwise fashion from ovarian epithelial inclusions, cystadenomas, and borderline tumors. The current study was designed to gain insight into the origins of low-grade serous carcinomas (tubal versus ovarian) by comparatively evaluating the morphologic (secretory and ciliated cell distribution) and immunophenotypic (using antibodies to PAX8, tubulin, calretinin, and Ki67) attributes of its putative precursor lesions, the normal tubal epithelium, and the overt malignancy. A total of 226 adnexal tissues from 178 patients were studied, including 98 adnexae removed for non-neoplastic indications, 48 serous cystadenomas, 42 serous borderline tumors, and 38 low-grade serous carcinomas. Normal distal tubal epithelium comprised an admixture of PAX8+/tubulin- secretory cells and PAX8-/tubulin+ ciliated cells with a proliferative index of ∼3%. The vast majority of ovarian surface epithelia displayed a mesothelial phenotype (calretinin+/PAX8-/tubulin-) and low proliferative index (0% (12 per 1000)), although 4% of cases also displayed foci with tubal phenotype (calretinin-/PAX8+/tubulin+). In contrast, most (78%) of the ovarian epithelial inclusions displayed a tubal phenotype and had a significantly higher proliferative index (1%) than ovarian surface epithelium, indicating that in most cases, the ovarian surface epithelium and ovarian epithelial inclusions are of different lineages. There was a progressive decrease in the population of ciliated cells, as evidenced by increasing secretory/ciliated cell ratio, from ovarian epithelial inclusions/cystadenomas to borderline tumors to low-grade serous carcinoma, indicating that the latter is a clonal expansion of secretory cells. Overall, the findings make a strong argument that the ovarian epithelial inclusions with a tubal phenotype is likely derived from fallopian tube through an intraovarian endosalpingiosis rather than through Mullerian metaplasia from ovarian surface epithelium. Genetic and molecular studies are needed to further confirm this finding as tubal origination of ovarian serous cancers will have a significant impact on ovarian cancer prevention and management.

Thyroid transcription factor 1 (NKX2-1/TITF1) mutations cause brain-lung-thyroid syndrome, characterized by congenital hypothyroidism (CH), infant respiratory distress syndrome (IRDS) and benign hereditary chorea (BHC). The objectives of the present study were (i) detection of NKX2-1 mutations in patients with CH associated with pneumopathy and/or BHC, (ii) functional analysis of new mutations in vitro and (iii) description of the phenotypic spectrum of brain-lung-thyroid syndrome. We identified three new heterozygous missense mutations (L176V, P202L, Q210P), a splice site mutation (376-2A->>G), and one deletion of NKX2-1 at 14q13. Functional analysis of the three missense mutations revealed loss of transactivation capacity on the human thyroglobulin enhancer/promoter. Interestingly, we showed that deficient transcriptional activity of NKX2-1-P202L was completely rescued by cotransfected PAX8-WT, whereas the synergistic effect was abolished by L176V and Q210P. The clinical spectrum of 6 own and 40 published patients with NKX2-1 mutations ranged from the complete triad of brain-lung-thyroid syndrome (50%), brain and thyroid disease (30%), to isolated BHC (13%). Thyroid morphology was normal (55%) and compensated hypothyroidism occurred in 61%. Lung disease occurred in 54% of patients (IRDS at term 76%; recurrent pulmonary infections 24%). On follow-up, 20% developed severe chronic interstitial lung disease, and 16% died. In conclusion, we describe five new NKX2.1 mutations with, for the first time, complete rescue by PAX8 of the deficient transactivating capacity in one case. Additionally, our review shows that the majority of affected patients display neurological and/or thyroidal problems and that, although less frequent, lung disease is responsible for a considerable mortality.

The ovary is a common site of involvement for metastasis and the breast is one of the most common sources. Metastatic breast carcinoma can mimic a primary ovarian carcinoma. Pax8 is a crucial transcription factor for organogenesis of the thyroid gland, kidney, and Müllerian system, and it also regulates Wilms tumor suppressor gene (WT1) expression. A total of 124 cases of ovarian carcinomas (84 serous papillary, 18 endometrioid, 12 mucinous, 10 clear cell) and 243 cases of invasive breast carcinomas (178 ductal, 65 lobular) were immunostained with Pax8 and WT1 by tissue microarrays to see the differential expression. Pax8 reaction was found in 108 of 124 ovarian carcinomas (87.1%) generally in diffuse staining, including 81 of 84 serous papillary carcinomas (96.4%), 16 of 18 endometrioid carcinomas (88.9%), 10 of 10 clear cell carcinomas (100%), and 1 of 12 mucinous carcinomas (8.3%), whereas WT1 expression was seen in 78 of 124 ovarian carcinomas (62.9%), including 73 of 84 serous papillary carcinomas (86.9%), and 5 of 18 endometrioid carcinomas (27.8%), with no expression in all 10 clear cell carcinomas and 12 mucinous carcinomas. All the mammary carcinomas were completely negative for Pax8, but WT1 expression was seen in 5 of 243 cases (2.1%). Pax8 is a useful marker in the differential diagnosis of ovarian and breast carcinomas, and it seems to be superior to WT1 for the diagnosis of all types of nonmucinous ovarian carcinomas, notably clear cell and endometrioid types where WT1 expression is generally negative or only focal.

Follicular thyroid carcinoma (FTC) and benign follicular adenoma (FA) are indistinguishable by preoperative diagnosis due to their similar histological features. Here we report the first RNA sequencing study of these tumors, with data for 30 minimally invasive FTCs (miFTCs) and 25 FAs. We also compared 77 classical papillary thyroid carcinomas (cPTCs) and 48 follicular variant of PTCs (FVPTCs) to observe the differences in their molecular properties. Mutations in H/K/NRAS, DICER1, EIF1AX, IDH1, PTEN, SOS1, and SPOP were identified in miFTC or FA. We identified a low frequency of fusion genes in miFTC (only one, PAX8-PPARG), but a high frequency of that in PTC (17.60%). The frequencies of BRAFV600E and H/K/NRAS mutations were substantially different in miFTC and cPTC, and those of FVPTC were intermediate between miFTC and cPTC. Gene expression analysis demonstrated three molecular subtypes regardless of their histological features, including Non-BRAF-Non-RAS (NBNR), as well as BRAF-like and RAS-like. The novel molecular subtype, NBNR, was associated with DICER1, EIF1AX, IDH1, PTEN, SOS1, SPOP, and PAX8-PPARG. The transcriptome of miFTC or encapsulated FVPTC was indistinguishable from that of FA, providing a molecular explanation for the similarly indolent behavior of these tumors. We identified upregulation of genes that are related to mitochondrial biogenesis including ESRRA and PPARGC1A in oncocytic follicular thyroid neoplasm. Arm-level copy number variations were correlated to histological and molecular characteristics. These results expanded the current molecular understanding of thyroid cancer and may lead to new diagnostic and therapeutic approaches to the disease.

Patients with germline mutation of succinate dehydrogenase (SDH) subunit genes are prone to develop paraganglioma, gastrointestinal stromal tumor, and rarely renal cell carcinoma (RCC). However, SDH-deficient RCC is not yet widely recognized. We identified such tumors by distinctive morphology and confirmed absence of immunohistochemical staining for SDHB. Immunohistochemical features were evaluated using a panel of antibodies to renal tumor antigens. Targeted next-generation sequencing was performed on DNA extracted from paraffin-embedded tissue. Eleven tumors were identified from 10 patients, 22-72 years of age (median 40). Two patients had paragangliomas, 1 bilateral SDH-deficient RCC, and 1 contralateral oncocytoma. Grossly, tumors were tan or red-brown, 2-20 cm in diameter (median 4.25 cm). Fuhrman grade was 2 (n=10) or 3 (n=1). Stage was pT1a-pT2b. One patient developed widespread metastases 16 years after nephrectomy and died of disease 6 years later. All tumors were composed of uniform eosinophilic cells containing vacuoles or flocculent cytoplasmic inclusions. Architecture was primarily solid; entrapped renal tubules and intratumoral mast cells were common. By immunohistochemistry, tumor cells were negative for SDHB (11/11) and rarely SDHA (1/11). Labeling was uniformly positive for PAX8 and kidney-specific cadherin and absent for KIT, RCC, and carbonic anhydrase IX. Staining for broad-spectrum epithelial markers was often negative or focal (positive staining for AE1/AE3 in 4/10, CAM5.2 3/7, CK7 1/11, EMA 10/10). By sequencing, SDHB mutation and loss of the second allele were present in 5/6 tumors; the SDHA-deficient tumor showed no SDHB abnormality. SDH-deficient RCC is a unique neoplasm that is capable of progression, often harboring SDHB mutation. A monomorphic oncocytic renal tumor with solid architecture, cytoplasmic inclusions of flocculent material, and intratumoral mast cells should prompt evaluation of SDH status, as it may have implications for screening the patient and relatives. Negative immunohistochemistry for KIT and heterogeneous labeling for epithelial antigens are other supportive features.

Cell-lineage-specific transcription factors are a group of regulatory proteins expressed in embryonic, differentiated, or neoplastic cells of the same lineage and represent a valuable repertoire of tissue-specific markers for the diagnosis of human tumors. Together with PAX2, PAX8 is a nephric-lineage transcription factor and is required for the establishment of renal-lineage cells and the formation of the kidney. In contrast to PAX2, little is known about the expression of PAX8 in adult kidney and renal tumors. In this study, we used immunohistochemistry to investigate the expression of PAX8 in adult human kidney and renal epithelial tumors. We report here that PAX8 was detected in renal epithelial cells in all segments of renal tubules from the proximal tubules to the renal papillae and in the parietal cells of Bowman's capsule in the adult kidney. PAX8 was also present in 98% of clear cell renal cell carcinomas (RCCs), 90% of papillary RCCs, and 95% of oncocytomas, similar to PAX2. In addition, PAX8 was found in 82% of chromophobe RCCs, 71% of sarcomatoid components of RCCs, and 100% (2/2) of renal medullary carcinomas. Overall, PAX8 was detected in 85% of metastatic renal tumors. Interestingly, expression of PAX8 was noted in some urothelial cells in the renal pelvis and ureters and approximately 23% of urothelial carcinomas of the renal pelvis, but not in the urothelium or urothelial carcinomas of the urinary bladder; this probably underlines the different embryonic origins of urothelial cells in the upper and lower urinary tracts. As shown in this study, PAX8 is widely expressed in normal and neoplastic renal tissues. PAX8 may be a useful additional marker for renal epithelial tumors; however, its specificity and sensitivity await further investigation.

OBJECTIVE

We used a single nucleotide polymorphism (SNP) map in a large cohort of 580 African American families to identify regions linked to type 2 diabetes, age of type 2 diabetes diagnosis, and BMI.

METHODS

After removing outliers and problematic samples, we conducted linkage analysis using 5,914 SNPs in 1,344 individuals from 530 families. Linkage analysis was conducted using variance components for type 2 diabetes, age of type 2 diabetes diagnosis, and BMI and nonparametric linkage analyses. Ordered subset analyses were conducted ranking on age of type 2 diabetes diagnosis, BMI, waist circumference, waist-to-hip ratio, and amount of European admixture. Admixture mapping was conducted using 4,486 markers not in linkage disequilibrium.

RESULTS

The strongest signal for type 2 diabetes (logarithm of odds [LOD] 4.53) was a broad peak on chromosome 2, with weaker linkage to age of type 2 diabetes diagnosis (LOD 1.82). Type 2 diabetes and age of type 2 diabetes diagnosis were linked to chromosome 13p (3-22 cM; LOD 2.42 and 2.46, respectively). Age of type 2 diabetes diagnosis was linked to 18p (66 cM; LOD 2.96). We replicated previous reports on chromosome 7p (79 cM; LOD 2.93). Ordered subset analysis did not overlap with linkage of unselected families. The best admixture score was on chromosome 12 (90 cM; P = 0.0003).

CONCLUSIONS

The linkage regions on chromosomes 7 (27-78 cM) and 18p overlap prior reports, whereas regions on 2p and 13p linkage are novel. Among potential candidate genes implicated are TCF7L1, VAMP5, VAMP8, CDK8, INSIG2, IPF1, PAX8, IL18R1, members of the IL1 and IL1 receptor families, and MAP4K4. These studies provide a complementary approach to genome-wide association scans to identify causative genes for African American diabetes.

OBJECTIVE

Genomic studies of ovarian cancer (OC) cell lines frequently used in research revealed that these cells do not fully represent high-grade serous ovarian cancer (HGSOC), the most common OC histologic type. However, OC lines that appear to genomically resemble HGSOC have not been extensively used and their growth characteristics in murine xenografts are essentially unknown.

METHODS

To better understand growth patterns and characteristics of HGSOC cell lines in vivo, CAOV3, COV362, KURAMOCHI, NIH-OVCAR3, OVCAR4, OVCAR5, OVCAR8, OVSAHO, OVKATE, SNU119 and UWB1.289 cells were assessed for tumor formation in nude mice. Cells were injected intraperitoneally (i.p.) or subcutaneously (s.c.) in female athymic nude mice and allowed to grow (maximum of 90 days) and tumor formation was analyzed. All tumors were sectioned and assessed using H&E staining and immunohistochemistry for p53, PAX8 and WT1 expression.

RESULTS

Six lines (OVCAR3, OVCAR4, OVCAR5, OVCAR8, CAOV3, and OVSAHO) formed i.p xenografts with HGSOC histology. OVKATE and COV362 formed s.c. tumors only. Rapid tumor formation was observed for OVCAR3, OVCAR5 and OVCAR8, but only OVCAR8 reliably formed ascites. Tumors derived from OVCAR3, OVCAR4, and OVKATE displayed papillary features. Of the 11 lines examined, three (Kuramochi, SNU119 and UWB1.289) were non-tumorigenic.

CONCLUSIONS

Our findings help further define which HGSOC cell models reliably generate tumors and/or ascites, critical information for preclinical drug development, validating in vitro findings, imaging and prevention studies by the OC research community.

Chronic sleep disturbances, associated with cardiometabolic diseases, psychiatric disorders and all-cause mortality, affect 25-30% of adults worldwide. Although environmental factors contribute substantially to self-reported habitual sleep duration and disruption, these traits are heritable and identification of the genes involved should improve understanding of sleep, mechanisms linking sleep to disease and development of new therapies. We report single- and multiple-trait genome-wide association analyses of self-reported sleep duration, insomnia symptoms and excessive daytime sleepiness in the UK Biobank (n = 112,586). We discover loci associated with insomnia symptoms (near MEIS1, TMEM132E, CYCL1 and TGFBI in females and WDR27 in males), excessive daytime sleepiness (near AR-OPHN1) and a composite sleep trait (near PATJ (INADL) and HCRTR2) and replicate a locus associated with sleep duration (at PAX8). We also observe genetic correlation between longer sleep duration and schizophrenia risk (rg = 0.29, P = 1.90 × 10-13) and between increased levels of excessive daytime sleepiness and increased measures for adiposity traits (body mass index (BMI): rg = 0.20, P = 3.12 × 10-9; waist circumference: rg = 0.20, P = 2.12 × 10-7).

BACKGROUND

Hurthle cell cancer (HCC) is an understudied cancer with poor prognosis.

OBJECTIVE

Our objective was to elucidate the genomic foundations of HCC.

METHODS

We conducted a large-scale integrated analysis of mutations, gene expression profiles, and copy number alterations in HCC at a single tertiary-care cancer institution.

METHODS

Mass spectrometry-based genotyping was used to interrogate hot spot point mutations in the most common thyroid oncogenes: BRAF, RET, NRAS, HRAS, KRAS, PIK3CA, MAP2K1, and AKT1. In addition, common oncogenic fusions of RET and NTRK1 as well as PAX8/PPARγ and AKAP9-BRAF were also assessed by RT-PCR. Global copy number changes and gene expression profiles were determined in the same tumor set as the mutational analyses.

RESULTS

We report that the mutational, transcriptional, and copy number profiles of HCC were distinct from those of papillary thyroid cancer and follicular thyroid cancer, indicating HCC to be a unique type of thyroid malignancy. Unsupervised hierarchical clustering of gene expression showed the 3 groups of Hurthle tumors (Hurthle cell adenoma [HA], minimally invasive Hurthle cell carcinoma [HMIN], and widely invasive Hurthle cell carcinoma [HWIDE] clustered separately with a marked difference between HWIDE and HA. Global copy number analysis also indicated distinct subgroups of tumors that may arise as HWIDE and HMIN. Molecular pathways that differentiate HA from HWIDE included the PIK3CA-Akt-mTOR and Wnt/β-catenin pathways, potentially providing a rationale for new targets for this type of malignancy.

CONCLUSIONS

Our data provide evidence that HCC may be a unique thyroid cancer distinct from papillary and follicular thyroid cancer.

Side population (SP) cells are characterized by their ability to efflux the vital dye Hoechst 33342 (Sigma-Aldrich, St. Louis, MO) due to expression of the ATP binding cassette (ABC)-dependent transporter ABCG2, and are highly enriched for stem/progenitor cell activity. In this study we identified SP cells in murine thyroid, which are composed of two populations of cells: CD45(-)/c-kit(-)/Sca1(+) and CD45(-)/c-kit(-)/Sca1(-) cells. Quantitative RT-PCR analysis revealed that SP cells highly express ABCG2 and the stem cell marker genes encoding nucleostemin and Oct4, whereas the expression of genes encoding the thyroid differentiation markers, thyroid peroxidase, thyroglobulin (TG), and TSH receptor, and two transcription factors, thyroid transcription factor 1 (TITF1) and paired PAX8, critical for thyroid specific gene expression, are low in SP cells as compared with the main population cells. In situ hybridization and double immunofluorescence demonstrated that cells expressing Abcg2 gene reside in the interfollicular space of the thyroid gland. Approximately half and a small percentage of the ABCG2-positive cells were also positive for vimentin and calcitonin, respectively. After 9 wk under three-dimensional thyroid primary culture conditions, main population cells formed an epithelial arrangement and follicle-like structures that are immunoreactive for TITF1 and TG. In contrast, SP cells demonstrated very few morphological changes without any epithelial or follicle-like structure and negative immunostaining for TITF1 and TG. These results demonstrate that thyroid possesses SP cells that may represent stem/progenitor cells.

Members of the Eyes absent (Eya) gene family are important for auditory system development. While mutations in human EYA4 cause late-onset deafness at the DFNA10 locus, mutations in human EYA1 cause branchio-oto-renal (BOR) syndrome. Inactivation of Eya1 in mice causes an early arrest of the inner ear development at the otocyst stage. To better understand the role of Eya1 in inner ear development, we analyzed the cellular and molecular basis of the early defect observed in the Eya1 mutant embryos. We report here that Eya1-/- otic epithelium shows reduced cell proliferation from E8.5 and increased cell apoptosis from E9.0, thus providing insights into the cellular basis of inner ear defect which occurred in the absence of Eya1. Previous studies have suggested that Pax, Eya and Six genes function in a parallel or independent pathway during inner ear development. However, it remains unknown whether Pax genes interact with Eya1 or Six1 during inner ear morphogenesis. To further evaluate whether Pax genes function in the Eya1-Six1 pathway or whether they interact with Eya1 or Six1 during inner ear morphogenesis, we have analyzed the expression pattern of Eya1, Pax2 and Pax8 on adjacent sections of otic epithelium from E8.5 to 9.5 by in situ hybridization and the inner ear gross structures of Pax2, Eya1 and Six1 compound mutants at E17.5 by latex paintfilling. Our data strongly suggest that Pax2 interacts with Eya1 during inner ear morphogenesis, and this interaction is critical for the development of all sensory areas in the inner ear. Furthermore, otic marker analysis in both Eya1-/- and Pax2-/- embryos indicates that Eya1 but not Pax2 regulates the establishment of regional specification of the otic vesicle. Together, these results show that, while Eya1 exerts an early function essential for normal growth and patterning of the otic epithelium, it also functionally synergizes with Pax2 during the morphogenesis of all sensory areas of mammalian inner ear.

Histone deacetylases (HDACs) regulate fundamental biological processes such as cellular proliferation, differentiation, and survival via genomic and nongenomic effects. This study examined the importance of HDAC activity in the regulation of gene expression and differentiation of the developing mouse kidney. Class I HDAC1-3 and class II HDAC4, -7, and -9 genes are developmentally regulated. Moreover, HDAC1-3 are highly expressed in nephron precursors. Short term treatment of cultured mouse embryonic kidneys with HDAC inhibitors (HDACi) induced global histone H3 and H4 hyperacetylation and H3K4 hypermethylation. However, genome-wide profiling revealed that the HDAC-regulated transcriptome is restricted and encompasses regulators of the cell cycle, Wnt/β-catenin, TGF-β/Smad, and PI3K-AKT pathways. Further analysis demonstrated that base-line expression of key developmental renal regulators, including Osr1, Eya1, Pax2/8, WT1, Gdnf, Wnt9b, Sfrp1/2, and Emx2, is dependent on intact HDAC activity. Treatment of cultured embryonic kidney cells with HDACi recapitulated these gene expression changes, and chromatin immunoprecipitation assays revealed that HDACi is associated with histone hyperacetylation of Pax2/Pax8, Gdnf, Sfrp1, and p21. Gene knockdown studies demonstrated that HDAC1 and HDAC2 play a redundant role in regulation of Pax2/8 and Sfrp1 but not Gdnf. Long term treatment of embryonic kidneys with HDACi impairs the ureteric bud branching morphogenesis program and provokes growth arrest and apoptosis. We conclude that HDAC activity is critical for normal embryonic kidney homeostasis, and we implicate class I HDACs in the regulation of early nephron gene expression, differentiation, and survival.

Perfluorooctanesulfonate (PFOS) is a persistent organic pollutant, the potential toxicity of which is causing great concern. In the present study, we employed zebrafish embryos to investigate the developmental toxicity of this compound. Four-hour post-fertilization (hpf) zebrafish embryos were exposed to 0.1, 0.5, 1, 3 and 5 mg/L PFOS. Hatching was delayed and hatching rates as well as larval survivorship were significantly reduced after the embryos were exposed to 1, 3 and 5 mg/L PFOS until 132 hpf. The fry displayed gross developmental malformations, including epiboly deformities, hypopigmentation, yolk sac edema, tail and heart malformations and spinal curvature upon exposure to PFOS concentrations of 1 mg/L or greater. Growth (body length) was significantly reduced in the 3 and 5 mg/L PFOS-treated groups. To test whether developmental malformation was mediated via apoptosis, flow cytometry analysis of DNA content, acridine orange staining and TUNEL assay was used. These techniques indicated that more apoptotic cells were present in the PFOS-treated embryos than in the control embryos. Certain genes related to cell apoptosis, p53 and Bax, were both significantly up-regulated upon exposure to all the concentrations tested. In addition, we investigated the effects of PFOS on marker genes related to early thyroid development (hhex and pax8) and genes regulating the balance of androgens and estrogens (cyp19a and cyp19b). For thyroid development, the expression of hhex was significantly up-regulated at all concentrations tested, whereas pax8 expression was significantly up-regulated only upon exposure to lower concentrations of PFOS (0.1, 0.5, 1 mg/L). The expression of cyp19a and of cyp19b was significantly down-regulated at all exposure concentrations. The overall results indicated that zebrafish embryos constitute a reliable model for testing the developmental toxicity of PFOS, and the gene expression patterns in the embryos were able to reveal some potential mechanisms of developmental toxicity.

By proposing TSH as a key negative regulator of bone turnover, recent studies in TSH receptor (TSHR) null mice challenged the established view that skeletal responses to disruption of the hypothalamic-pituitary-thyroid axis result from altered thyroid hormone (T(3)) action in bone. Importantly, this hypothesis does not explain the increased risk of osteoporosis in Graves' disease patients, in which circulating TSHR-stimulating antibodies are pathognomonic. To determine the relative importance of T(3) and TSH in bone, we compared the skeletal phenotypes of two mouse models of congenital hypothyroidism in which the normal reciprocal relationship between thyroid hormones and TSH was intact or disrupted. Pax8 null (Pax8(-/-)) mice have a 1900-fold increase in TSH and a normal TSHR, whereas hyt/hyt mice have a 2300-fold elevation of TSH but a nonfunctional TSHR. We reasoned these mice must display opposing skeletal phenotypes if TSH has a major role in bone, whereas they would be similar if thyroid hormone actions predominate. Pax8(-/-) and hyt/hyt mice both displayed delayed ossification, reduced cortical bone, a trabecular bone remodeling defect, and reduced bone mineralization, thus indicating that the skeletal abnormalities of congenital hypothyroidism are independent of TSH. Treatment of primary osteoblasts and osteoclasts with TSH or a TSHR-stimulating antibody failed to induce a cAMP response. Furthermore, TSH did not affect the differentiation or function of osteoblasts or osteoclasts in vitro. These data indicate the hypothalamic-pituitary-thyroid axis regulates skeletal development via the actions of T(3).